Compositions and methods for treatment of diseases involving cxcl1 function

ABSTRACT

The present disclosure relates to antibodies, for example monoclonal antibodies, and their use in clinical patient evaluation and therapy. The present disclosure further relates to a method for modulating the activity of human CXCL-1 protein (hereinafter, referred to as CXCL1). In an aspect, antibodies described herein are capable of being used as a medicament for the prevention and/or treatment of diseases involving CXCL1 function, for example, pathological angiogenesis and inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.16/982,456, filed Sep. 18, 2020, which is the National Stage entry ofInternational Application No. PCT/US20/15457, filed 28 Jan. 2020, whichclaims priority to U.S. Provisional Application No. U.S. 62/797,573,filed on Jan. 28, 2019. The content of the aforementioned applicationsare herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “3000047-002002_Sequence_Listing_ST25.txt” createdon 7 Mar. 2022, and 39,159 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

BACKGROUND 1. Field

The present disclosure relates to antibodies, for example monoclonalantibodies, and their use in clinical patient evaluation and therapy.The present disclosure further relates to a method for modulating theactivity of human chemokine (C-X-C motif) ligand 1, CXCL-1 protein(hereinafter, referred to as CXCL1). In an aspect, antibodies describedherein are capable of being used as a medicament for the preventionand/or treatment of diseases involving CXCL1 function, for example,pathological angiogenesis, inflammatory diseases and cancers.

2. Background

Angiogenesis is a process involving the growth of new blood vessels frompre-existing vessels. In healthy adults, angiogenesis is quiescent inmost of the organs, however, angiogenesis may occur in certainpathological conditions, such as during benign or malignant tumorgrowth, retinal disorders and wound healing. Targeting angiogenesis fortreating cancer was proposed in the 1970s and clinical studies haveresulted in the development of a number of therapeutic compounds,including humanized monoclonal antibodies to target this essentialaspect of tumor development. One humanized monoclonal antibody (mAb),bevacizumab that targets the angiogenic factor VEGF (VascularEndothelial Growth Factor) has obtained Food and Drug Agency (FDA)approval for treating cancers in association with standardchemotherapeutic agents. However, targeting VEGF alone has been onlypartially successful. Evasion of treatment could arise via variousmechanisms so there remains a need for additional anti-angiogeniccompounds that can be used alone or in combination with anti-cancertreatments, including other mAbs to improve clinical management andoutcomes for cancer patients.

Chemokines, also referred as to as chemotactic cytokines, are known tobe critical mediators of the inflammatory response by regulating therecruitment of cells from both the innate and adaptive immune systems todiseased tissues. Dysregulated expression and activity of certainchemokines have been implicated in cancer initiation and progression.Specifically, chronic chemokine exposure is associated with macrophageand T cell accumulation, chronic activation of macrophages, abnormalangiogenesis and DNA damage due to the presence of reactive oxygenspecies. Furthermore, chemokines have been known to regulate pivotalprocesses during tumor progression including primary tumor growth, tumorangiogenesis and development of metastatic disease. Chemokines may alsoenhance epithelial-stromal interactions facilitating tumor growth andinvasion.

For example, chemokines characterized by an ELR motif such as humanCXCL1/growth-related oncogene (GRO) is known to induce angiogenesis invitro and in vivo. CXCL1 binds to and activates the chemokine receptorCXCR2, which is considered the key receptor in the angiogenic reaction.CXCL1 is potently active on neutrophils, inducing a marked recruitmentof these cells into inflammatory sites. Neutrophils can synthesize andstore molecules with known angiogenic activity, including vascularendothelium growth factor (VEGF-A), CXCL8/IL-8, CXCL1/GRO, andhepatocyte growth factor, suggesting that neutrophils can play a keyrole in the angiogenic process induced by CXC-ELR⁺ chemokines includingCXCL1.

Human CXCL1 also functions as a tumor-related factor. It has been shownthat the amount of human CXCL1 fluctuates at the gene level and at theprotein level in the tissue or blood of patients with malignant tumors,such as urothelial cancer, large-bowel cancer, ovary cancer, ormalignant melanoma.

U.S. Pat. No. 9,309,312 discloses purified anti-human CXCL1 monoclonalantibodies or purified antigen-binding fragments thereof, whichspecifically recognize several amino acid sequence regions of CXCL1protein. U.S. Pat. No. 9,309,312 also discloses using these antibodiesor fragments in an immunoassay method for measuring a human CXCL1protein.

US 20160108117 discloses antibodies or fragments thereof directed toCXCL1, said antibody or fragment being capable of binding to the humanchemokine CXCL1 with an equilibrium dissociation constant (KD) of atmost 16 nM, as determined by surface plasmon resonance. US 20160108117also discloses using these antibodies or fragments in an in vitrodiagnostic and/or prognostic method of a pathological angiogenesisdisease or a disease characterized by undesirable excessiveneovascularization in a subject.

Advantageously, the novel materials and methods provided here forblocking CXCL1 overcome shortfalls of current angiogenesis inhibitordrugs. Anti-CXCL1 mAbs of the present disclosure could address theaforementioned limitations.

There remains a need to develop better methods and compositions that cantreat and/or prevent diseases involving pathological angiogenesis. Asolution to this technical problem is provided by the embodimentscharacterized in the claims.

BRIEF SUMMARY

In an aspect, the disclosure provides for anti-human chemokine CXCL1monoclonal antibodies or antigen-binding fragments thereof. In anotheraspect, the antibodies are isolated or recombinant.

In yet another aspect, the disclosure provides for anti-human chemokineCXCL1 monoclonal antibodies or antigen-binding fragments thereof whereinthe antibodies or the antigen-binding fragments thereof bind to humanchemokine CXCL1 protein having the amino acid sequence of

(SEQ ID NO: 5) ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN.

In another aspect, an antibody described herein comprises animmunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:2 or a variant thereof and an immunoglobulin light chain having theamino acid sequence of SEQ ID NO: 4 or a variant thereof.

The disclosure further provides for an antibody or the antigen-bindingfragment thereof, wherein the variant of the immunoglobulin heavy chaincomprises at least one amino acid addition, substitution, insertion,and/or deletion in the amino acid sequence of SEQ ID NO: 2. In anotheraspect, the variant of the immunoglobulin light chain comprises at leastone amino acid addition, substitution, insertion, and/or deletion in theamino acid sequence of SEQ ID NO: 4.

In another aspect, an antibody or antigen-binding fragment describedherein is produced by using hybridoma clone HL2401.

In an aspect, an antibody or antigen-binding fragment thereof describedherein is labeled with a toxin. In another aspect, an antibody orantigen-binding fragment thereof described herein is labeled with aradionucleotide, a fluorescent dye, a fluorescent protein, an enzyme,biotin and/or (strept)avidin. In yet another aspect, the radionucleotideis ⁶⁴Cu, ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P, or ³⁵S. In an aspect, thefluorescent dye is fluorescein isothiocyanate (FITC), rhodamine, Texasred, Cy3, or Cy5. In another aspect, the fluorescent protein isphycoerythrin (PE), allophycocyanin (APC), or green fluorescent protein(GFP). An enzyme described herein may be selected from the groupconsisting of horseradish peroxidase, alkaline phosphatase, or glucoseoxidase.

In an aspect, the disclosure provides for an isolated nucleic acidmolecule encoding an antibody or an antigen-binding fragment thereofdescribed herein.

The disclosure further provides for a vector comprising a nucleic acidmolecule described herein.

In an aspect, the disclosure further provides for a host cell comprisinga nucleic acid molecule or vector described herein.

The disclosure further provides for a pharmaceutical compositioncomprising at least one active ingredient selected from the groupconsisting of an antibody or an antigen-binding fragment thereofdescribed herein, a pharmaceutically acceptable carrier, and optionally,pharmaceutically acceptable excipient(s) and/or stabilizer(s).

The disclosure further provides for a method of treating a patient orindividual by administering a compound, composition, antibody, orantigen-binding fragment thereof to a patient or individual in needthereof. In an aspect, the patient or individual has a pathologicalangiogenesis disease or a disease caused by excessiveneovascularization. In another aspect, the pathological angiogenesisdisease or the disease caused by excessive neovascularization isselected from the group consisting of cancers with abnormalangiogenesis, ophthalmological diseases with abnormal angiogenesis,rheumatoid arthritis, psoriasis, angioma, endometriosis, and kaposisarcoma.

The disclosure further provides for a method of detecting human CXCL1 ina sample using an antibody or antigen-binding fragment thereof describedherein.

In another aspect, an antibody described herein comprises a heavy chainvariable domain comprising complementarity-determining region (CDR) 1consisting of the amino acid sequence of SEQ ID NO: 6, CDR2 consistingof the amino acid sequence of SEQ ID NO: 7, and CDR3 consisting of theamino acid sequence of SEQ ID NO: 8, and a light chain variable domaincomprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 9,CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and CDR3consisting of the amino acid sequence of SEQ ID NO: 11.

In another aspect, the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 12 or 34.

In another aspect, the heavy chain variable domain comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 14, 18,36, or 38.

In another aspect, the antibody comprises the amino acid sequenceselected from the group consisting of SEQ ID NO: 16, 20, 22, and 24.

In another aspect, the light chain variable domain comprises a signalpeptide comprising the amino acid sequence of SEQ ID NO: 26.

In another aspect, the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 28.

In another aspect, the heavy chain variable domain comprises a signalpeptide comprising the amino acid sequence of SEQ ID NO: 27.

In another aspect, the heavy chain variable domain comprises the aminoacid sequence of SEQ ID NO: 30 or 32.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows anti-CXCL1 neutralizing monoclonal mouse antibody (HL2401)binds to recombinant human CXCL1, but not mouse and rat CXCL1.

FIG. 1B shows the apparent affinity-binding constant (KD) value forCXCL1 in PBS and serum.

FIG. 1C shows immunoprecipitation followed by LC/MS-MS analysisconfirmed that HL2401 binds specifically to CXCL1.

FIG. 1D shows total RNA extraction using HL2401 hybridoma cell line.

FIG. 1E shows PCR amplification using HL2401 cDNA as template.

FIG. 1F shows numbering and regions the complementarity-determiningregions (CDRs) in the light chain (VL) of the HL2401 clone.

FIG. 1G shows numbering and regions the CDRs in the heavy chain (VH) ofthe HL2401 clone.

FIG. 1H shows homology modelling of mouse sequence using Rosettahomology modelling server.

FIG. 1I shows agarose gel electrophoresis of the amplified VH, VL andscFv fragments.

FIG. 1J shows Western blot analysis using HL2401 scFv protein.

FIG. 1K anti-Myc tag monoclonal antibody.

FIG. 1L shows ELISA signal that indicates HL2401_scFv proteininteracting with human CXCL1 protein.

FIG. 2A shows human cell lines T24, DU145 and PC3 express a range ofCXCL1 levels.

FIG. 2B shows CXCL1 protein is expressed in stably transfected cells.

FIG. 2C shows, in an in vitro migration and invasion assay, themigratory potential of stably transfected cells.

FIG. 2D shows, in a tube-formation assay, the total length of structuresformed by Human umbilical vein endothelial cells (HUVEC) affected bystably transfected cells.

FIG. 3A shows, in an in vitro proliferation assay, the effect of HL2401on proliferation of human cells.

FIG. 3B shows, in an in vitro invasion assay, the effect of HL2401 onthe invasive potential of human cell lines.

FIG. 3C shows the effect of HL2401 on HUVEC tube length.

FIG. 4A shows, following intraperitoneal administration, the plasmaconcentration of HL2401.

FIGS. 4B and 4C show, after a single injection of HL2401, theradiolabeled HL2401 antibody was rapidly distributed.

FIG. 4D shows the ex vivo bio-distribution data.

FIG. 5A shows no toxicity with HL2401 administration.

FIG. 5B shows immunofluorescent staining on the T24 and PC3 xenografttumors for CXCL1.

FIGS. 6A and 6B show apoptotic index in tumors from animals treated withHL2401.

FIG. 7 shows homology modelling of mouse and humanization templatesequence using Rosetta homology modelling server.

FIG. 8 shows homology modelling of mouse and humanization templatesequence using Rosetta homology modelling server.

FIG. 9 shows sequence alignment according to one embodiment of thepresent disclosure.

FIG. 10 shows sequence alignment according to another embodiment of thepresent disclosure.

FIG. 11A-11C shows the antigen CXCL1 and a negative control protein waselectrophoresed and transblotted to the nitrocellulose membrane.

FIG. 12 shows humanized version of HL2401_scFv protein expressed in E.coli and purified that was detected by anti-His tag antibody.

FIG. 13 shows the humanization version of H2407 scFvs format antibodyproteins are specially binding to human CXCL1 antigen in ELISA andWestern blot analysis.

FIG. 14 shows schematics for cloning of the light chain and heavy chainof humanized gene according to one embodiment of the present disclosure.

FIG. 15 shows ELISA results.

FIG. 16A-16C shows the antigen human CXCL1 and a negative controlbacterial cell lysate with chicken lysozyme protein were electrophoresedand transblotted to the nitrocellulose membrane.

FIGS. 17A and 17B show affinity purification chromatogram of purifiedsamples according to one embodiment of the present disclosure.

FIGS. 18A and 18B shows SDS-PAGE gel electrophoresis of purified samplesaccording to one embodiment of the present disclosure.

FIGS. 19A-19H show that humanized anti-human CXCL1 antibodies HumBB2401(a.k.a. BB2401) and Hum2401 are as effective as Avastin in theinhibition of endothelial cell sprouting.

FIGS. 20A-20F show that humanized anti-human CXCL1 antibodies HumBB2401(a.k.a. BB2401) and Hum2401 are as effective as Avastin in theinhibition of endothelial cell tube formation.

DETAILED DESCRIPTION

The present disclosure demonstrates the increased angiogenic potentialof human prostate cancer cells that over-expressed the Bcl2proto-oncogene. Specifically, increased Bcl2 expression enhanced thetumorigenic and angiogenic ability of human prostate cancer xenografts.For example, culturing human endothelial cells (HUVEC and HDMEC) inconditioned media from Bcl2-overexpressing human prostate cancer cellsresulted in increased rates of proliferation and the expression of keyanti-apoptotic genes/proteins. This possibly provides a survivaladvantage over endothelial cells grown in conditioned media from cancercells with low Bcl2 expression. Comparative genomic profiling of thetreated and untreated endothelial cells revealed approximately 250differentially expressed genes (p<0.001). After validation studies,CXCL1, a chemokine, stood out among several secreted proteins ofinterest (fold-change 3.96, p<2.22E-16).

CXCL1, a secreted growth factor that interacts with theG-protein-coupled receptor CXCR2, plays an important role not only inangiogenesis but also in inflammation and is a known chemo-attractantfor neutrophils. Through a series of investigations, the presentinventors showed that CXCL1 influences neo-angiogenesis throughregulation of EGF and ERK 1/2 signaling, and cellular proliferation viaincreases in cyclin D3 and cdk4 levels, and have confirmed the role ofCXCL1 in tumor establishment and survival in in vitro and in vivostudies.

Given the role of CXCL1 in angiogenesis, it is possible to inhibitangiogenesis, thus treat diseases, by blocking the binding of CXCL1 toCXCR2. Therefore, pharmaceutical agents blocking the CXCL1 pathway arecapable of treating many angiogenesis-dependent diseases, including butnot limited to, cancer.

Monoclonal antibodies (mAbs) have become a new class of therapeuticagents due to their ability to bind with high-specificity to a target,their long plasma half-life, and their low toxicity/side effects.Furthermore, with the advent of full human antibody technology,immunogenicity issues are avoided. Therefore, monoclonal antibodies havebecome a mainstay for pharmaceutical compositions.

Currently, no mAb containing amino acid sequence identical or similar tothe present disclosure has been disclosed for the inhibition ofangiogenesis and the treatment of cancers and otherangiogenesis-dependent diseases.

The present disclosure provides mAb that specifically bind to CXCL1. TheCXCL1 mAb can not only bind but also neutralize CXCL1.

In one embodiment, the present disclosure describes antibodies, orportions thereof, binding to CXCL1.

In another embodiment, the antibodies can be used to block angiogenesis,including treating angiogenesis-dependent diseases. Such disordersinclude, but are not limited to, retinopathy, age related maculardegeneration, chronic articular rheumatism and psoriasis, disordersassociated with inappropriate or inopportune invasion of vessels such asdiabetic retinopathy, neovascular glaucoma, restenosis, capillaryproliferation in atherosclerotic plaques and osteoporosis, and cancerassociated disorders, such as solid tumors, solid tumor metastases,angiofibromas, retrolental fibroplasia, hemangiomas, Kaposi's sarcomaand all cancers which may require neovascularization to support tumorgrowth.

In another embodiment, the present disclosure also provides nucleicacids comprising nucleotide sequences encoding such antibodies; vectorscomprising such nucleic acids; host cells and organisms comprising suchnucleic acids and/or vectors; and compositions, such as pharmaceuticallyacceptable compositions and kits, comprising such proteins, nucleicacids, vectors, and/or cells and typically one or more additionalingredients that can be active ingredients or inactive ingredients thatpromote formulation, delivery, stability, or other characteristics ofthe composition (e.g., various carriers).

In another embodiment, the present disclosure also provides amino acidcomprising amino acid sequences encoding such antibodies.

In another embodiment, the present disclosure further provides uses ofantibodies or fragments thereof in the modulation of CXCL1-mediatedbiological activities, for example, inhibiting vascular endothelial cellproliferation and angiogenesis in the treatment ofangiogenesis-dependent diseases related thereto.

Advantageously, the materials and methods provided herein for blockingbinding of CXCL1 to its cognate receptor, i.e., CXCR2, overcomeshortfalls of current angiogenesis inhibitor drugs. The antibodies ofthe present disclosure target an independent angiogenic pathwaydifferent from that of the classical VEGF/VEGFR pathway. The CXCL1chemokine pathway is both angiogenic and inflammatory. Moreover, theCXCL1 chemokine can induce an autocrine proliferation pathway becausetumor cells express the receptors CXCR1 and CXCR2. Thus, by inhibitingthe CXCL1 chemokine, the antibodies or fragments thereof of the presentdisclosure may inhibit angiogenesis, inflammation, and proliferation.

In another embodiment, the mAbs according to the present disclosure maybe used as a medicament. In particular, the mAbs can be used for thetreatment of angiogenesis-dependent diseases.

The term “human CXCL1” as used herein refers to a protein or a naturalmutant thereof comprising the amino acid sequence according to GenbankNM_001511. The term “natural mutant” refers to a mutant existing in thenature. Examples of such a mutant include a mutant comprising an aminoacid sequence having a deletion, a substitution, an addition, or aninsertion of one or several amino acids in the aforementioned amino acidsequence of human CXCL1 and a mutant having 95% or more, preferably 98%or more, and more preferably 99% or more amino acid sequence identitywith the aforementioned amino acid sequence of human CXCL1. Here, theterm “identity” refers to the percentage (%) of the total number ofamino acid residues of the amino acid sequence in question that areidentical to amino acid residues of the amino acid sequence of humanCXCL1 when the two amino acid sequences are aligned such that thehighest possible degree of agreement between them is achieved. In thiscase, sequence alignment can be carried out by introducing or notintroducing gaps, and the number of gaps introduced is included when thepercentage is calculated. Also, the term “several” refers to an integerbetween 2 and 10, such as between 2 and 7, 2 and 5, 2 and 4, and 2 and3. Specific examples of a natural mutant include mutants based onpolymorphism such as SNP (single nucleotide polymorphism) and splicingmutants. The above substitution is preferably a conservative amino acidsubstitution. If the substitution is a conservative amino acidsubstitution, a mutant resulting from the conservative amino acidsubstitution may have a structure or properties substantially equivalentto those of human CXCL1 having the above amino acid sequence. Asconservative amino acids, nonpolar amino acids (glycine, alanine,phenylalanine, valine, leucine, isoleucine, methionine, proline, andtryptophan) and polar amino acids (amino acids other than nonpolar aminoacids), charged amino acids (acidic amino acids (aspartic acid andglutamic acid) and basic amino acids (arginine, histidine, and lysine))and non-charged amino acids (amino acids other than charged aminoacids), aromatic amino acids (phenylalanine, tryptophan, and tyrosine),branched amino acids (leucine, isoleucine, and valine), and aliphaticamino acids (glycine, alanine, leucine, isoleucine, and valine), areknown, for example.

In an embodiment, conservative substitutions may include those, whichare described by Dayhoff in “The Atlas of Protein Sequence andStructure. Vol. 5”, Natl. Biomedical Research, the contents of which areincorporated by reference in their entirety. For example, in an aspect,amino acids, which belong to one of the following groups, can beexchanged for one another, thus, constituting a conservative exchange:Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine(S), threonine (T); Group 2: cysteine (C), serine (S), tyrosine (Y),threonine (T); Group 3: valine (V), isoleucine (I), leucine (L),methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K),arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y),tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamicacid (E). In an aspect, a conservative amino acid substitution may beselected from the following of T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G,and/or T→S.

In a further embodiment, a conservative amino acid substitution mayinclude the substitution of an amino acid by another amino acid of thesame class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met,Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3)acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative aminoacid substitutions may also be made as follows: (1) aromatic: Phe, Tyr,His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) protonacceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Pat.No. 10,106,805, the contents of which are incorporated by reference intheir entirety).

In another embodiment, conservative substitutions may be made inaccordance with Table 1. Methods for predicting tolerance to proteinmodification may be found in, for example, Guo et al., Proc. Natl. Acad.Sci., USA, 101(25):9205-9210 (2004), the contents of which areincorporated by reference in their entirety.

In an aspect, sequences described herein may include 1, 2, 3, 4, 5, 10,15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions,deletions.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multi-specific antibodies (e.g., bi-specific antibodies),and antibody fragments, so long as they exhibit the desired biologicalactivity. Various techniques relevant to the production of antibodiesare provided in Harlow et al. Antibodies: A laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1988.

The term “monoclonal antibody” as used herein refers to a polypeptidecontaining an immunoglobulin- or its fragment-derived framework region(FR) and a complementarity determining region (CDR) and being capable ofspecifically binding to and recognizing an antigen. Therefore, the term“anti-human CXCL1 monoclonal antibody” in the present disclosure refersto a polypeptide capable of specifically binding to human CXCL1 or afragment thereof and recognizing the human CXCL1 or a fragment thereof.The term “specifically binding” refers to binding to only a targetantigen (human CXCL1 or a fragment thereof in the present disclosure).

A typical immunoglobulin molecule consists of a tetramer in which twosets, each consisting of two polypeptide chains referred to as a heavychain and a light chain, are connected to each other via disulfide bond.A heavy chain comprises a heavy chain variable region (VH) on theN-terminus and a heavy chain constant region (CH) on the C-terminus. Alight chain comprises a light chain variable region (VL) on theN-terminus and a light chain constant region (CL) on the C-terminus. Ofthese regions, VH and VL are particularly important since they areinvolved in the binding specificity of the antibody. VH and VL eachcomprises about 110 amino acid residues, wherein three complementaritydetermining regions (CDR1, CDR2, and CDR3) directly involved in bindingspecificity with an antigen and four framework regions (FR1, FR2, FR3,and FR4) functioning as framework structures for variable regions arepresent. A complementary determining region is known to formconformation complementary to an antigen molecule and determine thespecificity of the relevant antibody (E. A. Kabat et al., 1991,Sequences of proteins of immunological interest, Vol. 1, eds. 5, NIHpublication). Whereas amino acid sequences of constant regions remainalmost unchanged among antibodies of the same species, amino acidsequences of complementary determining regions are highly variable amongantibodies. Hence, complementary strand determining regions are alsoreferred to as hypervariable regions. In a variable region, suchcomplementarity determining regions (CDRs) and framework regions arearranged in the direction from an amino acid terminus to a carboxyterminus in order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. VL and VHform a dimer with each other so as to form an antigen binding sitewithin an immunoglobulin molecule. Regarding immunoglobulin, IgG, IgM,IgA, IgE, and IgD classes are known. The antibody of the presentdisclosure may be of any class and is preferably IgG.

An antibody useful in the present disclosure may be derived from everyanimal source including birds and mammals. Examples of such the animalor bird source include mice, rats, guinea pigs, rabbits, goats, donkeys,sheep, camels, horses, chickens, and humans. Also, “monoclonal antibody”in the present disclosure may be chemically synthesized or synthesizedusing a recombinant DNA method. For example, recombinant antibodies suchas chimeric antibodies and humanized antibodies are also encompassed inthe present disclosure.

A “humanized” antibody is a human/non-human chimeric antibody thatcontains minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which resides from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such a mouse, rat, rabbit having the desiredspecificity, affinity and capacity.

The term “hypervariable” region when used herein refers to the aminoacid residues of an antibody that are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR”” (residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domainand/or those residues from a ‘hypervariable loop” (residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain.

The term “fragment thereof” in “monoclonal antibody or a fragmentthereof” as used herein refers to a partial region of the antibody andspecifically refers to a polypeptide chain or a complex thereof havingactivity substantially equivalent to the antigen-specific bindingactivity of the antibody. Examples of such a fragment include anantibody portion containing at least one of the above antigen bindingsites and specifically, a polypeptide chain or a complex thereof havingat least one VL and at least one VH. Specific examples of such apolypeptide chain or a complex thereof include many sufficientlycharacterized antibody fragments and the like generated via cleavage ofimmunoglobulin with various peptidases. More specific examples of suchantibody fragments include Fab, F(ab′)2, and Fab′. Fab is a fragmentgenerated by cleaving an IgG molecule with papain, by which cleavage iscarried out at a position closer to the N-terminal side than thedisulfide linkage of a hinge part. Fab is composed of a polypeptidecomprising VH and CH1 which is adjacent to VH among the 3 domains (CH1,CH2, and CH3) composing CH and a light chain. F(ab′)2 is a dimer ofFab′, which is generated by cleaving an IgG molecule with pepsin at aposition closer to the C-terminal side than the disulfide linkage of thehinge part. Fab′ has a structure substantially equivalent to that ofFab, although the H chain is somewhat longer than that of Fab since itcontains the hinge part (Fundamental Immunology, Paul ed., 3d ed.,1993). Fab′ can be obtained by reducing F(ab′)2 under mild conditionsand then cleaving the disulfide linkage in the hinge region. All ofthese antibody fragments contain antigen binding sites, so that they arecapable of specifically binding to antigens (that is, human CXCL1 or afragment thereof in the present disclosure).

The above “fragment thereof” in the present disclosure may be chemicallysynthesized or synthesized using a recombinant DNA method. An example ofsuch a fragment is an antibody fragment newly synthesized using arecombinant DNA method. Specific examples of such a fragment include,but are not limited to, a monomeric polypeptide molecule prepared byartificially linking one or more VL and one or more VH of the antibodyof the present disclosure via a linker peptide or the like having anappropriate length and sequence and a multimeric polypeptide thereof.Examples of such a polypeptide include single chain Fv (scFv: singlechain fragment of variable region) (see Pierce catalog and Handbook,1994-1995, Pierce Chemical co., Rockford, Ill.) and synthetic antibodiessuch as a diabody, a triabody, and a tetrabody. In an immunoglobulinmolecule, VL and VH are generally separately located on differentpolypeptide chains (a light chain and a heavy chain). Single chain Fv isa synthetic antibody fragment that has a structure in which thesevariable regions are linked with a flexible linker having a sufficientlength and the linked regions are contained in a single polypeptidechain. Within single chain Fv, both variable regions can beself-assembled to form a single functional antigen binding site. Singlechain Fv can be obtained by incorporating a recombinant DNA encoding thesingle chain Fv into a phage genome using a known technique and thencausing the expression of the DNA. A diabody is a molecule having astructure based on the dimeric structure of single chain Fv (Holliger etal., 1993, Proc. Natl. Acad. Sci. U.S.A., 90: 6444-6448). For example,when the length of the above linker is shorter than about 12 amino acidresidues, two variable sites within single chain Fv cannot undergoself-assembly. However, the two variable sites are caused to form adiabody and specifically two single chain Fvs are caused to interactwith each other, enabling the assembling of VL of one Fv chain and VH ofthe other Fv chain. Hence, two functional antigen binding sites can beformed (Marvin et al., 2005, Acta Pharmacol. Sin., 26: 649-658).Moreover, a cysteine residue is added to the C-terminus of single chainFv, so that disulfide bond of the two Fv chains can be formed andthereby formation of a stable diabody become possible (Olafsen et al,2004, Prot. Engr. Des. Sel., 17: 21-27). As described above, a diabodyis a divalent antibody fragment. However, each antigen binding site isnot required to bind to the same epitope and may have bi-specificitysuch that the antigen binding sites recognize and specifically bind todifferent epitopes. A triabody and a tetrabody have a trimeric structureand a tetrameric structure, respectively, based on a single chain Fvstructure in a manner similar to a diabody. A triabody and a tetrabodyare a trivalent antibody fragment and a quadrivalent antibody fragment,respectively, or may be multiple specific antibodies.

Furthermore, examples of the above “fragment thereof” include antibodyfragments that are identified using phage display libraries (e.g., seeMcCafferty et al., 1990, Nature, Vol. 348, 522-554) and haveantigen-binding capacity. In addition, also see Kuby, J., Immunology,3rd ed., 1998, W. H. Freeman & Co., New York, for example.

The antibody or a fragment thereof of the present disclosure can bemodified. The term “modified or modification” used herein refers to bothfunctional modification required for the antibody or a fragment thereofof the present disclosure to have activity of specifically binding tohuman CXCL1 (e.g., glycosylation) and modification for labeling requiredfor detection of the antibody or a fragment thereof of the presentdisclosure. In one embodiment, antibodies or fragments thereof bind tothe extracellular domains of two or more targets of a protein selectedfrom the group consisting of the above-mentioned proteins, and theaffinity value (Kd) is less than 1×10 μM. Also, glycosylation of theantibody of the present disclosure may be altered for adjusting theaffinity of an antibody for a target antigen. Such alteration can beachieved by, for example, changing one or more glycosylation siteswithin the antibody sequence. More specifically, for example, one ormore amino acid substitutions are introduced into an amino acid sequencecomposing one or more glycosylation sites within FR so as to remove theglycosylation sites, so that deglycosylation can be achieved at thesites. Such deglycosylation is effective for increasing the affinity ofan antibody for an antigen (U.S. Pat. No. 5,714,350 and U.S. Pat. No.6,350,861). Optionally, the antibody carries a further effector functionsuch as an immune stimulating domain or toxin.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The present disclosure contemplates pharmaceutical (or therapeutic)composition useful for practicing the therapeutic methods describedherein. Therapeutic compositions of the present disclosure contain aphysiologically tolerable carrier together with a therapeuticallyeffective amount of an antibody as described herein, dissolved ordispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic or has reducedimmunogenicity when administered to a mammal or human patient fortherapeutic purposes. A therapeutically effective amount is an amount ofan antibody of the disclosure sufficient to produce a measurableinhibition of angiogenesis in the tissue being treated, i.e., anangiogenesis-inhibiting amount. Inhibition of angiogenesis can bemeasured in situ by immunohistochemistry, or by other methods known toone skilled in the art.

The pharmaceutical compositions may contain the antibodies either in thefree form or in the form of a pharmaceutically acceptable salt. As usedherein, “a pharmaceutically acceptable salt” refers to a derivative ofthe disclosed antibodies wherein the antibodies may be modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, ptoluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on antibodies may be prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike. In an especially preferred embodiment, the pharmaceuticalcompositions comprise the antibodies as salts of acetic acid (acetates),trifluoro acetates or hydrochloric acid (chlorides).

Pharmaceutical composition according to the disclosure may containadjuvant selected from the group consisting of colony-stimulatingfactors, such as Granulocyte Macrophage Colony Stimulating Factor(GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, andinterferon-alpha. In a preferred embodiment, the pharmaceuticalcomposition according to the disclosure the adjuvant is selected fromthe group consisting of colony-stimulating factors, such as GranulocyteMacrophage Colony Stimulating Factor (GM-CSF, sargramostim),cyclophosphamide, imiquimod and resiquimod. In a preferred embodiment ofthe pharmaceutical composition according to the disclosure, the adjuvantis cyclophosphamide, imiquimod or resiquimod. Even more preferredadjuvants are Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V,Montanide ISA-51, poly-ICLC (Hitonol®) and anti-CD40 mAB, orcombinations thereof.

The medicament of the disclosure may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CDS-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present disclosure. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-17 4, OM-197-MPEC, ONTAK, OspA, PepTek® vector system,poly(lactide co-glycolide) [PLG]-based and dextran microparticles,talactoferrin SRL 172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including antibodiesor protein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 81 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present disclosure. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivatives thereof (e.g. AmpliGen®, Hiltonol®,poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA aswell as immunoactive small molecules and antibodies such ascyclophosphamide, sunitinib, Bevacizumab®, celebrex, NCX-4016,sildenafil, tadalafil, vardenafil, sorafenib, temozolomide,temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171,anti-CTLA4, other antibodies targeting key structures of the immunesystem (e.g., anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present disclosure can readily be determined by theskilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivatives, poly-(1:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes. In apreferred embodiment, the pharmaceutical composition according to thedisclosure the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe disclosure the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the disclosure, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the antibodies and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Theantibodies can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

Method for Preparing Monoclonal Antibody and Hybridoma

One embodiment of the present disclosure provides a neutralizing IgG mAbto CXCL1. Specifically, mAb to CXCL1 may be prepared by first immunizingan appropriate animal, such as mouse, with CXCL1 protein. Hybridomas arethen produced and screened for the production of CXCL1-reactive IgGantibodies using standard techniques (e.g., ELISA).

In order to generate a fully human anti-CXCL1 antibody, the DNAsequences of the heavy (SEQ ID NO: 1) and light (SEQ ID NO: 2) chains ofthis antibody were obtained by PCR using cDNA that had beenreverse-transcribed from the RNA of antibody hybridoma as templates. Asis well known in the art, individual amino acids can be encoded bydifferent DNA sequences. Hence, the amino acid sequences of thisantibody can be encoded by different DNA sequences, e.g., SEQ ID. NO: 1encoding the amino acid sequence of the heavy chain (SEQ ID. NO: 2) andSEQ ID. NO: 3 encoding the amino acid sequence of the light chain (SEQID. NO: 4). These DNA sequences fall within the scope of the presentdisclosure. Furthermore, based on the common knowledge of antibodystructure, some amino acids in an antibody may be substituted, deleted,or added, without detracting the biological activities of the antibody.In some cases, changes in the amino acid sequence of an antibody mayeven improve the biological activities and/or improve certain propertiescompared to the original antibody. Therefore, it is possible to modifythe amino acid sequences of this anti-CXCL1 antibody to obtain antibodyvariants with similar, or even improved, biochemical or biologicalproperties. These modified antibodies are within the scope of thepresent disclosure.

Anti-CXCL1 mAb of the present disclosure can be “humanized” throughgenetic engineering techniques known to those of ordinary skills in theart. After selecting high-affinity anti-CXCL1 IgG antibody-producingclones (e.g., HL2401) that are capable of neutralizing CXCL1, the cellsare genetically engineered so that the hypervariable regions from anon-human antibody combining site derived from an anti-CXCL1 antibodycan be ‘grafted’ onto the framework regions of human IgG antibody. Thistechnique is known as complementary-determining region (CDR) grafting,which provides the production of a humanized IgG antibody having apre-selected non-human antibody binding site specific for a givenepitope on CXCL1. These IgG antibodies are then mass produced in HEK293cells and then purified.

Similar to the above, humanized CXCL1 mAb was screened; a) for theproduction of CXCL1-reactive IgG antibodies using standard techniques(e.g., ELISA), b) for inhibition of in vitro tube formation and c) forinhibition of interaction between CXCL1 and CXCR2.

Antibodies of the disclosure are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of the pharmaceuticallyacceptable carrier include saline, Ringer's solution and dextrosesolution. The pH of the solution is preferably from about 5 to about 8,and more preferably from about 7 to about 7.5. Further carriers includesustained release preparations such as semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, liposomes or microparticles. Itwill be apparent to those persons skilled in the art that certaincarriers may be more preferable depending upon, for instance, the routeof administration and concentration of antibody being administered. Theantibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of antibodies, preferably for treating retinopathy, agerelated macular degeneration, chronic articular rheumatism andpsoriasis, disorders associated with inappropriate or inopportuneinvasion of vessels such as diabetic retinopathy, neovascular glaucoma,restenosis, capillary proliferation in atherosclerotic plaques andosteoporosis, and cancer associated disorders, such as solid tumors,solid tumor metastases, angiofibromas, retrolental fibroplasia,hemangiomas, Kaposi's sarcoma and all cancers which requireneovascularization to support tumor growth, the efficacy of thetherapeutic antibody can be assessed in various ways well known to theskilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

One embodiment of the present disclosure provides a method for detectionof CXCL1 in a sample using the antibodies or fragments thereof describedherein. In an aspect, the CXCL1 is human CXCL1. In particular, thepresent disclosure provides a method for detecting human CXCL1 in asample, comprising the step of incubating the sample with the antibodyand/or fragment thereof according to the invention.

In some embodiments, the sample is a biological sample.

The term “biological sample” encompasses a variety of sample typesobtained from an organism that may be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen, or tissuecultures or cells derived there from and the progeny thereof.Additionally, the term “biological sample” may encompass circulatingtumor or other cells. The term “biological sample” specificallyencompasses a clinical sample, and further includes cells in cellculture, cell supernatants, cell lysates, serum, plasma, urine, amnioticfluid, biological fluids including aqueous humour and vitreous for eyesamples, and tissue samples. The term “biological sample” alsoencompasses samples that have been manipulated in any way afterprocurement, such as treatment with reagents, solubilization, orenrichment for certain components.

In some embodiments, the biological sample can be selected from a bodilyfluid, a fraction thereof, a tissue extract, and a cell extract. Inparticular, the biological sample can be selected from a plasma sampleand a tumor extract.

In some embodiments, the method for detecting human CXCL1 in a samplefurther comprises the step of detecting the binding of the antibody orfragment thereof according to the invention to human CXCL1.

The method for detecting human CXCL1 in a sample according to theinvention can be based on various techniques, well known by one skilledin the art, including, but not limited to:

a western blot assay (CXCL1 or fragment thereof present in a cell lysateor in a solution being immobilized on a membrane, the membrane beingthereafter incubated with the antibody of the invention, preferablylabeled, in appropriate conditions well known in the art),

an ELISA assay (CXCL1 or fragment thereof being immobilized on amicrotiter plate, the said plate being thereafter incubated with theantibody of the invention, preferably labeled, in appropriate conditionswell known in the art),

an immunohistochemistry assay (the antibody, preferably labeled, beingused to stain a sample containing fixed cells or tissues expressingCXCL1 or a fragment thereof, in appropriate conditions well known in theart),

a flow cytometry assay (the antibody, preferably labeled, being used tostain a sample containing fixed or living cells expressing CXCL1 or afragment thereof, in appropriate conditions well known in the art),

In one embodiment of the method for detecting human CXCL1 in a sampleaccording to the invention, the antibody of the invention is coated on asolid support.

These detection techniques are described, for example, in Sambrook,Fritsch and Maniatis—“Molecular Cloning A Laboratory Manual SecondEdition Cold Spring Harbor Laboratory, 1989, the content of which isincorporated by reference in its entirety. Any other detectiontechniques requiring the use of an antibody are herein encompassed. Thepresence and/or the amount of human CXCL1 in the sample can bedetermined by one or more of these techniques. Some of these techniquesrequire labeling the antibody of the invention with a detectable marker,preferably a fluorescent or a luminescent marker.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multifunctional and bedetectable by more than one of the methods listed. These antibodies maybe directly or indirectly labeled with said probes. Attachment of probesto the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ. Examples of the above antibodylabels may include fluorescent dyes, e.g., fluorescein isothiocyanate(FITC), rhodamine, Texas red, Cy3, and Cy5, fluorescent proteins, e.g.,phycoerythrin (PE), allophycocyanin (APC), and green fluorescent protein(GFP), enzymes, e.g., horseradish peroxidase, alkaline phosphatase, andglucose oxidase, and biotin or (strept)avidin. The antibody may belabeled with a radionucleotide, such as ⁶⁴Cu, ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I,³H, ³²P or ³⁵S, so that the tumor can be localized usingimmunoscintiography.

The present disclosure also provides a method for purifying human CXCL1from a sample, comprising the step of incubating the sample with theantibody and/or fragment thereof according to the invention.

The method for purifying human CXCL1 from a sample according to theinvention can be based on various techniques, well known by one skilledin the art, including, but not limited to flow cytometry assays,immunoprecipitation assays. These detection techniques are described,for example, in Sambrook, Fritsch and Maniatis—“Molecular Cloning ALaboratory Manual Second Edition Cold Spring Harbor Laboratory, 1989.

The present disclosure also provides a kit useful for carrying out themethods for detecting and/or purifying human CXCL1 in a sample accordingto the invention, the kit comprising: at least one antibody or fragmentthereof according to the invention; and at least one reagent fordetecting the antibody or fragment thereof according to the invention.

The reagent for detecting the antibody or fragment thereof according tothe invention can be selected from the group consisting of ELISAreagents, Western blot reagents, and dot blot reagents.

The present disclosure also provides for in vitro or ex vitro diagnosticand/or prognostic method, and particularly to an in vitro diagnosticand/or prognostic method of a disease related to CXCL1, particularly ofa pathological angiogenesis disease, in particular of clear cell renalcell carcinoma, in a subject, the method comprising: determining theexpression and/or the level of expression of CXCL1 in a biologicalsample of the subject using at least one antibody and/or fragmentthereof according to the invention.

Treatment of Pathologic Angiogenesis

The present disclosure provides for a method for the inhibition ofangiogenesis in tissues, and thereby inhibiting events in the tissues,which depend upon angiogenesis. Generally, the method comprisesadministering to the tissue a composition comprising anangiogenesis-inhibiting amount of an antibody of the present disclosure.

As described earlier, angiogenesis includes a variety of processesinvolving neovascularization of a tissue including “sprouting”,vasculogenesis, or vessel enlargement, all of which angiogenesisprocesses involve disruption of extracellular matrix collagen in bloodvessels. With the exception of traumatic wound healing, corpus leuteumformation and embryogenesis, it is believed that the majority ofangiogenesis processes are associated with disease processes andtherefore the use of the present therapeutic methods is selective forthe disease.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,fibroplasia, hemangiomas, Kaposi's sarcoma and the like cancers whichrequire neovascularization to support tumor growth. Other suitabletumors include melanoma, carcinoma, sarcoma, fibrosarcoma, glioma andastrocytoma. Thus, methods, which inhibit angiogenesis in a diseasedtissue, ameliorate symptoms of the disease and, depending upon thedisease, can contribute to cure of the disease.

In some aspects, the antibodies or the antigen-binding fragment thereofof the disclosure are administered alone or in combination withanti-cancer treatments, such as, for example one or morechemotherapeutic agent, one or more immunotherapeutic agent, orcombinations thereof.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.Tissue, as used herein, also encompasses all bodily fluids, secretionsand the like, such as serum, blood, cerebrospinal fluid, plasma, urine,synovial fluid, vitreous tumor.

Representative Routes of Administration

The antibodies of the disclosure can be administered parentally byinjection or by gradual infusion over time. Although the tissue to betreated can typically be accessed in the body by systemic administrationand therefore most often treated by intravenous administration oftherapeutic compositions, other tissues and delivery means arecontemplated where there is a likelihood that the tissue targetedcontains the target molecule. Thus, antibodies and derivatives, thereofcan be administered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracaviatary, intravesically, transdermally,topically, intraocually, orally, or intranasally.

The description herein of any aspect or embodiment of the presentdisclosure using terms such as “comprising”, “having”, “including” or“containing” with reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the disclosurethat “consists of', “consists essentially of', or “substantiallycomprises” that particular element or elements, unless otherwise statedor clearly contradicted by context (e.g., a composition described hereinas comprising a particular element should be understood as alsodescribing a composition consisting of that element, unless otherwisestated or clearly contradicted by context).

EXAMPLES

The present disclosure may be further demonstrated through the followingexamples. It should be understood that the scope of this disclosure isnot limited to the examples. Furthermore, those with skill in the artmay modify or alter this disclosure after reading this disclosure; thesemodified variants should be regarded as equivalent to embodiments andfall into the scope of this disclosure.

The following examples, if not described in detail, use techniquescommonly known to those with skill in the art and may follow theexperimental protocols or conditions described by references such asMolecular Cloning, A Laboratory Manual (Sambrook, etc., Cold SpringHarbor Laboratory Press) or Antibodies: A Laboratory Manual, (Ed Harlowand David Lane, Cold Spring Harbor Laboratory Press), etc., or based onmanufacture's instruction.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples, which illustrate procedures for practicing thedisclosure. These examples should not be construed as limiting.

Example 1 Preparation of Human Anti-CXCL1 Antibody

Balb/cByJ mice were immunized multiple times with human recombinantCXCL1 protein. The anti-CXCL1 antibody titers in mouse sera weredetermined by the ELISA assay. After high anti-CXCL1 antibody titer inserum was reached, spleens were dissected and splenocytes isolated tofuse with myeloma cells to generate hybridoma cells. The fusedhybridomas were grown in selection medium to generate hybridoma clones.

A mouse monoclonal antibody against CXCL1 was produced using a standardprotocol of the Hybridoma and Protein Core Laboratories, University ofFlorida Interdisciplinary Center for Biotechnology Research (ICBR)(Chang et al, 2013). Two female Balb/cByJ mice were immunized withapproximately 100 μg of native CXCL1 protein having the amino acidsequence ofASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN (SEQ ID NO: 5) diluted in sterile physiologic phosphatebuffered saline (PBS) and emulsified in Ribi MPL+TDM adjuvant. Theimmunogen was administered on days 1, 21, 44, and 192. The test bleedswere collected 11 to 14 days after the second and third immunizations.The presence of anti-CXCL1 antibodies in the post-immunized serum wasdetermined by western blots and ELISA. Six days after the fourthimmunization, mouse #1 was euthanized and the splenic lymphocytes werecollected and fused with mouse myeloma cells to form hybridoma cells(Uehara et al, 2005). The cultured media of the growing hybridoma masscultures were collected and screened for anti-CXCL1 antibody productionby ELISA. The mass cultures that tested positive by ELISA weresubsequently tested for biologic effect in a proliferation assayutilizing HUVEC cells. The cultures that showed reactivity to CXCL1 inELISA and exhibited anti-proliferative effects were grown out, andfurther cloned by limiting dilution. The cultured media collected fromeach clone were tested again by ELISA. The monoclonal antibodies wereisotyped by ELISA and IsoStrip tested following manufacturer's protocol.The cultured medium of the final selected hybridoma clone was harvested,and purified through a protein G column (GE Healthcare Protein GSepharose 4 Fast Flow). The concentration of the purified monoclonalanti-CXCL1 antibody (HL2401) was determined by Bradford Protein Assayand stored at 4° C. for future validation. A gel clot LAL assay fromLonza (Basel, Switzerland) ensured the antibody was free of endotoxins.

The hybridoma cell clones were grown in 96-well plates in RPMI-1640complete medium. Supernatants were collected from each hybridoma cloneand assayed for specific antibodies using ELISA. In this assay, ELISAplates were coated with soluble recombinant human CXCL1 and blocked with2% BSA. Then, hybridoma supernatants were properly diluted and added toeach well, followed by HRP-conjugated goat anti-mouse IgG. The plateswere then incubated with HRP substrate and OD values read at awavelength of 650 nm using a microplate reader.

A number of hybridoma clones that secrete anti-CXCL1 antibodies wereidentified. The hybridoma clones that secrete specific antibodiesagainst CXCL1 were expanded to 6-well plates, and then T-175 flasks.Supernatants were harvested from the flasks. Isotypes of the antibodiessecreted from hybridoma clones were determined using an IgG isotypingkit and concentrations of antibodies in the supernatants were measuredby an ELISA assay using the corresponding antibody subtypes as astandard. Antibody concentration in the hybridoma supernatants werenormalized and diluted. The antibody supernatants were used to comparerelative binding affinities of antibodies to CXCL1 by an ELISA assay. Bythis approach, several monoclonal antibodies clones that show highaffinity to CXCL1 were identified.

After the hybridoma clones expressing anti-CXCL1 antibodies wereidentified in the initial screening and inhibition of tube formation insecondary screen, the ability of the antibodies to block CXCL1-CXCR2binding were examined in a tertiary screen. In this tertiary screeningassay, 96-well ELISA plates were coated with human recombinant CXCL1 andblocked with BSA. In a separate 96-well plate, anti-CXCL1 antibodies atvarious concentrations were mixed with recombinant human CXCR2. Themixtures were incubated at 37° C. for one hour and then transferred tothe ELISA plate that was blocked with BSA. After rinse, HRP-conjugatedgoat anti-human IgG was added to each well, followed by HRP substrateand OD reading at 650 in a microplate reader. By this methodology, amonoclonal antibody clone, designated as HL2401 that is capable ofcompletely blocking CXCL1-CXCR2 binding was identified. HL2401 wasconfirmed as IgG by antibody isotyping.

FIG. 1A shows anti-CXCL1 neutralizing monoclonal mouse antibody (HL2401)binds to recombinant human CXCL1, but not mouse and rat CXCL1. Incontrast, a commercial antibody shows cross reactions with mouse, rat,and human CXCL1. This result shows the superior specificity of HL2401 tothat of the commercial antibody.

FIG. 1B shows the apparent affinity-binding constant (K_(D)) value forCXCL1 in PBS and serum (1:4 dilution and 1:40 dilution) were 175.6±30.0ng/mL, 386.6±75.4 ng/and 252.2±37.1 ng/mL, respectively.

FIG. 1C shows immunoprecipitation followed by LC/MS-MS analysisconfirmed that HL2401 binds specifically to CXCL1. That is, CXCL1 isonly immunoprecipitated with HL2401 and not with the control antibody,e.g., IgG.

Amino Acid Sequence of the mAb Variable Region

Total RNA was prepared from hybridoma cells using RNA extraction kit.The synthesize cDNA using SuperScript® III One-Step RT-PCR System(Invitrogen™) according to the manufacturer's instructions. The cDNA wasthen used as the template for PCR by using set of primers designed aspreviously described (Yuan et al. 2004). The PCR products were purifiedusing the Qiagen PCR clean up system (Qiagen) and ligated into pCRTM2.1vector. The selected positive clones were sequenced using the BigDyeTerminator v3.1 Cycle Sequencing kit (Applied Bio-systems). The aminoacid sequences were determined from the nucleotide sequence using IMGTand designated CDRs of light and heavy chain of isolated immunoglobulingenes.

FIG. 1D shows total RNA extraction using HL2401 hybridoma cell line.

FIG. 1 E shows PCR amplification using HL2401 cDNA as template. Lane 1and 2 indicated variable heavy chain and Lane 3 and 4 indicated variablelight chain. The light chain lanes represented segments of aberrantpseudogene PCR products.

TABLE 1 Summary of CDRs denotation of HL2401 hybridoma cell HL2401Heavy chain Light chain clone (VH) (VL) CDR1 SYYIY KASQSVDY (SEQ IDDGDSYVN NO: 6) (SEQ ID NO: 9) CDR2 EIDPSHG AASNLES GPTFN (SEQ ID (SEQ IDNO: 10) NO: 7) CDR3 TRESGTG QQSSEDPWT AMDY (SEQ ID (SEQ ID NO: 11)NO: 8)Amino acid sequences were deduced from DNA sequences. CDRs were selectedas described according to kabat numbering.

FIG. 1F shows numbering & regions the CDRs in the light chain (VL) ofthe HL2401 clone.

FIG. 1G shows numbering & regions the CDRs in the heavy chain (VH) ofthe HL2401 clone.

Classification of Variable Region Genes

Variable region of heavy chain Result summary: Productive IGH rearrangedsequence: V-GENE and allele Musmus IGHV1S81*02 J-GENE and allele MusmusIGHJ4*01 F

Variable region of light chain Result summary: Productive IGK rearrangedsequence: V-GENE and allele Musmus IGKV3-4*01 F J-GENE and allele MusmusIGKJ1*01 F

The light chain variable region of the HL2401 antibody gene belonged tothe immunoglobulin mouse kappa, V_(K)III (IG_(K)V₃) subgroup andcontained J_(K1) gene segments. The heavy chain belonged to theimmunoglobulin mouse V_(H) I (IGVH₁) subgroup gene family with J_(H4)segments.

FIG. 1H shows homology modelling of mouse sequence using Rosettahomology modelling server. The diagram was generated using PyMolemolecular graphic system. VH represents for variable heavy chain and VLrepresents for variable light chain.

Construction of scFv and test biological activity against Human CXCL-1antigen

The clone of variable heavy chain and light chain of HL2401 antibodygene were amplified using mouse primers. The amplified VH and VL domainwere purified using PCR purification Kit. The resultant VH and VLfragments were overlapping using pull through PCR and amplified as scFv(Single chain Fragment variable region). The gene encoding the scFv isVH-linker-VL with a standard 20 amino acid linker (Gly4Ser) 3 GGGAR. Theamplified gene was digested with BssHII and NheI restriction enzymes andinsert into a pET-based vector (PAB-myc) containing a pelB promotor forcontrolling periplasmic protein expression (Novagen, Madison, Wis.)along with 6×histidine tag at the C-termini for purification by metalaffinity chromatography and transformed into DH5α bacterial strain. Thetransformed clones were amplified in LB with ampicillin broth overnight.The plasmids DNA were prepared and sent for DNA sequencing. The correctsequence of scFv plasmid was transform T7 Shuffle bacterial strain andthe transformed bacteria were used for soluble protein production.

FIG. 1I shows agarose gel electrophoresis of the amplified VH, VL andscFv fragments (VH: variable heavy chain; VL: variable light chain;ScFv: single chain fragment variable region).

Induction of ScFv Proteins in Bacterial Host

The HL2401_scFv clone was transformed into T7 shuffle bacterial strain.T7 shuffle cells and was grown in 1.4 L 2×YT plus ampicillin medium at37° C. until log-phage (OD600=0.5), induced with 0.3 mM IPTG, andallowed to grow at 30° C. for an additional 16 hrs. After induction, thebacteria were harvested by centrifugation at 8000 g for 15 min at 4° C.,and the pellets were stored in −20° C. for at least 2 hrs. The frozenpellets were briefly thawed and suspended in 40 ml of lysis buffer (1mg/ml lysozyme in PBS plus EDTA-free protease inhibitor cocktail (ThermoScientific, Waltham, Mass.). The lysis mixture was incubated on ice foran hour, and then 10 mM MgCL2 and 1 ug/ml DNase I were added and themixture was incubated at 25° C. for 20 min. The final lysis mixture wascentrifuged at 12000 g for 20 min and the supernatants were collected.This supernatant was termed the periplasmic extract used for Nicklecolumn affinity chromatography.

Western Blots Analysis Using HL2401 scFv Protein

Purified recombinant human CXCL1 protein was used as antigen target inWestern blot analyses. 500 ng human CXCL1 protein and 1 μg purifiedprotein as negative control were loaded onto 4-20% gradient Tris-glycineSDS-PAGE and transferred onto nitrocelluler membranes. The membrane wasblocked using 3% skimmed milk in PBS for 3 h at room temperature. Afterthat, the membrane was incubated with partial purified HL2401_scFvprotein overnight at 4° C. The membrane was washed with sodium phosphatebuffered saline with 0.05% tween 20 buffer (PBST) 3 times. The washedmembrane was incubated with anti-c Myc mouse IgG for 1 h at roomtemperature to recognize the c-Myc tag on the scFv and identify theposition of antigens bound by the scFv. After washing with PBST, themembrane was incubated with the goat anti-mouse IgG (H+L) HRP conjugatediluted (1:3000 v/v) in PBS for 1 h at RT, and specific immunoreactivebands were visualized with a mixture of TMB substrate.

FIG. 1J shows Western blot analysis using HL2401 scFv protein. Lane Mindicates molecular weight markers. Lane 1 indicates human CXCL1protein. Lane 2 indicates negative control.

Western blot analysis detected approximately 11 kDa band using TMBstained. In addition, this Western blot data confirmed antibodyspecificity to target protein. On the other hand, as shown in FIG. 1 K,an anti-Myc tag monoclonal antibody was used to recognize the Myc tag onthe expressed of scFv protein. The antigen loaded membrane was incubatedwith anti-myc-HRP ( 1/2000) antibody and specific immunoreactive bandswere visualized with a mixture of TMB substrate. HL2401_scFv proteinexpressed in E. coli and partially purified that was detected byanti-cMyc antibody.

ELISA Test for Confirm the Binding Activity of HL2401_scFv Protein

The human CXCL1 protein was coated onto 96-well, 30 ng for well at 4° C.overnight. The plate was blocked by 3% skim milk in PBS 2 h at roomtemperature. The plate was washed with PBST and applied anti-human CXCL1scFv antibody at different dilution concentration. The anti-Myc mousemonoclonal antibody with HRP conjugate antibody was applied anddeveloped with TMB solution.

FIG. 1L shows ELISA signal that indicates HL2401_scFv proteininteracting with human CXCL1 protein.

Example 2

FIGS. 2C, 2D, and 3A-3C show CXCL1 expression stimulates cellproliferation, cellular migration and invasion and endothelial tubeformation.

To determine the effect of CXCL1 on key tumor cell and endothelial cellprocesses, human cell lines T24, DU145 and PC3 were first tested fortheir expression levels of CXCL1 and its receptor, CXCR2.

FIG. 2A shows bladder cancer cell line (T24) and prostate cancer cellline (PC3) express high levels of CXCL1. On the other hand, CXCR2, thereceptor for CXCL1, was highly expressed in T24 cells.

To test the effect of CXCL1 expression on cellular functions, tumor celllines, e.g., DU145, which do not express detectable CXCL1, were stablytransfected with CXCL1 to generate CXCL1-expressing tumor cells. On theother hand, tumor cell lines, e.g., T24 and PC3 cells, which highlyexpress CXCL1, were stably transfected with CXCL1-targeting shRNAvectors to knockdown their endogenous CXCL1 expression. Plasmids withsequence verified human CXCL1 cDNA cloned within pCMV6-Empty vector andplasmid with vector alone (Origene Technologies) were transfected intoDU145 cells using Fugene HD transfection reagent (Roche Diagnostics) tocreate DU145-CXCL1 and DU145-Empty. Similarly, CXCL1 short hairpin RNA(shRNA) cloned within pRS vector was transfected into T24 and PC3 cellsas well as CXCL1 plasmid scramble (Scr) non-effective shRNA constructwithin pRS vector (Origene) using Fugene HD. Stable transfectants wereselected with 1,200 μg/ml of G418 (Life Technologies, Inc., Carlsbad,Calif.) for DU145 clones and 0.25 μg/ml of puromycin (Life Technologies)for T24 and PC3 clones for 14 days and subcloned by limiting dilution in96-well plates. Integration of the transfected gene into the genome wasconfirmed by RT-PCR. Stable cell lines were maintained in mediacontaining 500 μg/ml of G418 for DU145 clones and in media containing0.25 μg/ml of puromycin for T24 and PC3 clones.

FIG. 2B shows reduced CXCL1 protein expression is confirmed in celllines stably transfected with CXCL1-targeting shRNA vectors, i.e.,T24-CXCL1-KD4 and T24-CXCL1-KD8; and PC3-CXCL1-KD7. On the other hand,CXCL1 expression is confirmed in DU145 cells stably transfected withCXCL1 (DU145-CXCL1-OE3 and DU145-CXCL1-OE8).

To determine the effect of CXCL1 on tumor cell migration or invasion, anin vitro migration and invasion assay was performed. Migration assayswere performed in 6 well two-tier invasion chambers (CollaborativeBiomedical Products, Bedford, Mass., USA) (Gomes Giacoia et al, 2014).Polycarbonate membranes were coated with 4 mg/mL growth factor reducedMatrigel (BD Biosciences, San Jose, Calif.) as described for invasionassays, control inserts (migration only) contained no coating. Twoseparate experimental designs were tested. First, DU145-CXCL1-OE3 andDU145-CXCL1-OE 8, DU145-Empty, T24-CXCL1-KD4 and T24-CXCL1-KD8,T24-shSCR, PC3-CXCL1-KD7, and PC3-shSCR cells were added to each insertat a density of 10⁵ cells/ml/well in RPMI media. The lower chambercontained RPMI media with 10% FBS as a chemoattractant. The cells weremaintained in a humidified incubator in 5% CO₂ at 37° C. for 24 hours.After the designated time, the cells on the top of the polycarbonatemembrane were removed. The cells attached to the bottom of the membranewere stained for 1 hour with cell viability indicator Calcein AMFluorescent Dye (BD Biosciences, Franklin Lakes, N.J.) and quantifiedusing the FLUOstar OPTIMA at 495 mm excitation and 515 nm emission (BMGLABTECH Inc., Cary, N.C.).

FIG. 2C shows, in an in vitro migration and invasion assay, themigratory potential of DU145-CXCL1-OE3 and DU145-CXCL1-OE8 clones wasnot enhanced compared to the DU145-Empty control. However, the invasivepotential of DU145-CXCL1-OE3 and DU145-CXCL1-OE8 clones wassignificantly enhanced by at least 50% compared to DU145-Empty (p<0.01).Similarly, the migratory potential of PC3-CXCL1-KD7 cells was notreduced compared to PC3-shSCR, but the invasive potential wassignificantly reduced by 27% in PC3-CXCL1-KD7 compared to PC3-shSCR(p<0.01). The same phenomenon was also observed in the migration andinvasion assays of T24 clones. Specifically, T24-CXCL1-KD8 cells(p<0.01) but not T24-CXCL1-KD4 cells showed an inhibition in cellmigration, however both T24-CXCL1-KD4 and T24-CXCL1-KD8 clonesdemonstrated a significant inhibition (at least 25%) of invasivepotential compared to T24-shSCR (p<0.01). These results suggest thatCXCL1 expression may play an important role in tumor cell invasion and,possibly, tumor cell migration.

To test the effect of CXCL1 on endothelial cell behaviour, a capillarytube formation assay was performed. Human umbilical vein endothelialcell (HUVEC) tube formation assay is one of the most widely used invitro model in angiogenesis research. HUVEC cells express CXCR2 andundergo cell proliferation and sprouting in response to CXCL1stimulation. Briefly, Matrigel (BD Biosciences) was added to 96-wellplates (50 μl per well) and allowed to solidify for 30 min at 37° C.HUVEC cells were incubated in serum- and growth factor-free EBM2 basalmedia containing 0.1% delipidated BSA for 5 hrs. HUVECs were seeded ontop of Matrigel in triplicates at a density of 10⁴ cells per well inconditioned media and incubated for 6 hrs. Images were acquired with aNikon ECLIPS E400 microscope (Nikon, Melville, N.Y.). The total lengthof tube-like structures in at least 4 viewed fields per well wasmeasured using ImageJ. At least three independent experiments consistingof each condition tested in triplicate wells was used to calculatemean±SD values.

HUVEC cultures were treated with conditioned media from the cell linesshown in FIG. 2B. FIG. 2D shows, in a tube-formation assay, the totallength of structures formed by HUVECs on growth factor reduced Matrigelwas significantly enhanced (˜60%) when treated with media fromDU145-CXCL1-OE3 and DU145-CXCL1-OE8 clones. In contrast, the totallength of tube-like structures was significantly reduced when treatedwith conditioned media from CXCL1-knockdown T24 (T24-CXCL1-KD4 andT24-CXCL1-KD8) and PC3 (PC3-CXCL1-KD7) cells (˜50% and ˜28%,respectively). These results suggest that CXCL1 may promote angiogenesisby inducing endothelial cell tube formation.

Example 3 Targeting CXCL1 Inhibits Proliferation, Cellular Invasion andEndothelial Tube Formation

To test whether CXCL1 inhibitors, such as anti-CXCL1 neutralizingmonoclonal mouse antibody (HL2401), could affect proliferation, a cellproliferation assay was performed. Briefly, 10³ cells (T24, DU145, andPC3) per well were plated in 96-microwell plates and incubated for 6,24, 48 and 72 hours with the indicated concentration of HL2401 for 72hrs. Each condition was tested in triplicate wells. Cell proliferationwas determined by incorporation of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Atleast three independent experiments were performed in triplicate.

FIG. 3A shows, in an in vitro proliferation assay at 72 hours,proliferation of T24, PC3, and HUVEC cell lines, but not DU145 cells,were significantly inhibited by HL2401 (20 and 100 μg/mL). Theanti-CXCL1 mAb can completely block CXCL1-induced HUVEC proliferationand sprouting.

To test whether anti-CXCL1 neutralizing monoclonal mouse antibody(HL2401) could affect tumor cell invasion, T24, DU145 and PC3 cells (10⁵cells/mL/well) were exposed to 0-200 μg/ml of CXCL1 monoclonal antibody(HL2401) in RPMI media. The lower chamber contained RPMI media with 10%FBS as chemoattractant. After 24 hours, the T24, DU145 and PC3 cells onthe top of the polycarbonate membrane were removed, while T24, DU145 andPC3 cells attached to the bottom of the membrane were stained for 1 hourwith cell viability indicator Calcein AM Fluorscent Dye and quantifiedusing the FLUOstar OPTIMA. For the migration and invasion assays, atleast three independent experiments consisting of each condition testedin triplicate wells was used to calculate mean±SD values.

FIG. 3B shows, in an in vitro invasion assay, the invasive potential ofT24 and PC3 was significantly reduced with the addition of HL2401 (20μg/mL) (p<0.01). DU145 invasive potential was unchanged by the additionof HL2401. These results suggest CXCL1 inhibitors, such as HL2401, caninhibit invasion of tumor cells that express CXCL1.

To demonstrate anti-angiogenic effects of this anti-CXCL1 antibody, theantibody was evaluated in an in vitro HUVEC tube formation assay.Briefly, HUVEC cells were seeded into 96-well plates coated withMatrigel. After 30 minutes, cells previously fed with serum and growthfactor free EBM2 basal medium for 5 hours were plated with the abovemedia supplemented with 0, 20 or 100 ug/ml of CXCL1 mAb (HL2401). After6 hours, photographic images of each well were obtained. Images wereacquired with a Nikon ECLIPS E400 microscope (Nikon, Melville, N.Y.).The total lengths of the tube structures were recorded. The total lengthof tube-like structures in at least 4 viewed fields per well wasmeasured using ImageJ. At least three independent experiments consistingof each condition tested in triplicate wells was used to calculatemean±SD values.

FIG. 3C shows that in the wells that contain 20 μg/ml of anti-CXCL1 mAb(HL2401), HUVEC tube length was significantly reduced and all sproutingwas inhibited at 100 μg/ml. In contrast, in the control wells in whichthe same amount (1 μg/ml) of normal antibody was added, no inhibitoryeffect on HUVEC tube formation was observed. Hence, the anti-CXCL1antibody of the present disclosure can completely inhibit vascularendothelial cell proliferation and sprouting; thus, this antibody iscapable of blocking angiogenesis. In a further study, it was shown thatthe HL2401 antibody does not bind to mouse CXCL1. Therefore, it is notsuitable to assess the biological activity of this antibody in regularmouse in vivo models.

Example 4 Pharmacokinetic Studies and Bio-Distribution

To determine the effect on HL2401 by in vivo administration,pharmacokinetics studies were performed in female C57BL/6 mice todetermine plasma exposure to CXCL1 antibody after single administration.CXCL1 antibody was radiolabeled. Briefly, ⁶⁴Cu was produced with anonsite cyclotron (GE PETrace). ⁶⁴CuCl₂ (74 MBq) was diluted in 300 μL of0.1 M sodium acetate buffer (pH 5.5) and mixed with 200 μL of NOTA-CXCL1antibody (0.5 mg/mL). The reaction was conducted at 37° C. for 45 minwith constant shaking. The resulting ⁶⁴Cu-NOTA-CXCL1 antibody waspurified by PD-10 size exclusion column chromatography, using PBS as themobile phase. The radioactive fraction containing ⁶⁴Cu-NOTA-CXCL1antibody was collected for in vivo studies.

Plasma samples at the following time points post-injection were taken:time zero (no treatment), 12, 24 and 48 hours. Plasma was derived fromthe whole blood by centrifugation at 3,000 rpm at 4° C. in plasmaseparator tubes for 10 minutes. All samples were stored at −80° C. untilsubsequent analysis. Samples were analyzed for CXCL1 antibody using anindirect ELISA. The lower limit of quantifications was 0.94 ng/mL inplasma. Pharamcokinetics parameters were calculated usingnoncompartmental analysis in WinNonLin v 5.0.3.

At different time points post-injection (p.i.) of 5-10 MBq of64Cu-NOTA-CXCL1 antibody via tail vein, PET scans of ICR mice (Envigo,Indianapolis, Ind.; n=4) were carried out using a microPET/microCTInveon rodent model scanner (Siemens Medical Solutions USA, Inc.). Dataacquisition, image reconstruction, and region-of-interest (ROI) analysisof the PET data were performed. Briefly, the images were acquired by 40million-count static PET scans and reconstructed using maximum aposteriori (MAP) algorithm, without attenuation or scatter correction.ROI analysis of each PET scan was carried out using software (InveonResearch Workplace, IRW) based on decay-corrected whole-body images,calculated with the injected dose measured by a dose calibrator(Capintec, Inc., Ramsey, N.J.). Quantitative PET data of the tumor andmajor organs was presented in the format of percentage injected dose pergram of tissue (% ID/g). After the last scan at 48 h p.i.,biodistribution studies were performed to corroborate PET data. Micewere euthanized and blood and major organs/tissues were collected andwet-weighed. The radioactivity in the tissue was measured using a γcounter (Perkin-Elmer, Norwalk, Conn.) and presented as % ID/g(mean±SD).

FIG. 4A shows, following intraperitoneal administration, the plasmaconcentration of HL2401 declined rapidly, due to rapid distribution toperipheral components. Limitations of assay sensitivity preventedcharacterization of terminal elimination (i.e., excretion).Concentration time analysis of HL2401 in plasma after a single dose of 4mg/kg or 8 mg/kg was 22.89 ng/g and 46.71 ng/g (C_(max)), 2.49 hours and2.71 hours (t_(1/2)) and 0.046 units and 0.044 units (clearance),respectively.

Similarly, FIGS. 4B and 4C show, after a single injection of 0.5 mg/kg,the radiolabeled antibody was rapidly distributed, remaining above thelimits of detection for over 48 hours on PET imaging (C_(max)=19.15% D/gat 15 min, t_(1/2α)≈3.5 min, t_(1/2β)≈44.0 hours).

FIG. 4D shows the ex vivo bio-distribution data that is well matchedwith the imaging results, thus, confirming the accuracy of PET imaging.

Example 5 Inhibition of Tumor Growth by HL2401 in Xenograft Model

The importance of CXCL1 expression for tumorigenicity and angiogenesiswas assessed in vivo using bladder cancer (T24) and prostate cancer(PC3) mouse xenograft models. DU145 xenografts were not generatedbecause DU145 cells do not express CXCL1 or CXCR2. As such, HL2401 maygenerate minimal therapeutic response in DU145 xenografts. To determinewhether targeting CXCL1 with a monoclonal antibody could inhibitxenograft tumor growth, CXCL1 antibody HL2401 was administered In vivo.Animal care was in compliance with the recommendations of The Guide forCare and Use of Laboratory Animals (National Research Council) andapproved by University of Hawaii local IACUC. Subcutaneoustumorigenicity assay was performed in athymic BALB/c nu/nu male mice (6to 8 weeks old) purchased from Envigo by inoculating 2×10⁶ parental T24cells and 2×10⁶ parental PC3 cells, as described previously (Miyake etal, 2015; Sakai et al, 2009b). After one week, mice bearingsubcutaneously xenograft tumors were divided randomly into three groups(Control, 4 mg/kg or 8 mg/kg) of HL2401 and treatment was initiated.Each group contains at least 10 mice. No toxicity or weight loss wasnoted in any of the treatment groups. HL2401 (100 μl diluted in sterilePBS) was administered via intraperitoneal injection twice weekly forfour weeks. Control mice received IgG alone on the same schedule. Tumorvolumes were measured weekly with digital calipers and calculated by V(mm³)=length×(width)²×0.5236. After five weeks of cell inoculation, themice were sacrificed, tumors resected and analyzed byimmunohistochemical staining.

FIG. 5A shows no toxicity (i.e., no weight change or activity change) inmice treated with HL2401 (8 mg/kg). At the end of 5 wk endpoint of an invivo study, control T24 xenografts reached an average of 388 mm³ insize. T24 xenografts treated twice weekly with 4 mg/kg of HL2401 reached274 mm³ (p=0.22) and only 224 mm³ when treated with 8 mg/kg (p<0.05).Similarly, PC3 tumors in mice treated with 4 mg/kg and 8 mg/kg of HL2401were reduced (p=0.15 and p<0.05, respectively) in size at theexperimental endpoint (only 8 mg/kg of HL2401 data shown).

FIG. 5B shows immunofluorescent staining on the T24 and PC3 xenografttumors for CXCL1 and PECAM-1 to indicate the location of CXCL1. In bothT24 and PC3 xenografts, CXCL1 was expressed in the tumor cells inaddition to the tumor-associated endothelial cells. IHC analysis ofexcised xenografts revealed a reduction in CXCL1 expression when treatedwith 8 mg/kg of HL2401. CXCR2 expression was more prevalent in T24xenografts compared to PC3 xenografts and CXCR2 expression levels inthese tumors treated with HL2401. Furthermore, a reduction ofinterleukin 6 (IL-6) and an increase in metalloproteinase inhibitor 4(TIMP4) are shown in both T24 and PC3 tumors from animals treated with 8mg/kg of HL2401. This result is consistent with the data of anangiogenesis PCR array, in which 84 targets was queried from twoindependent experiments and significant fold change deviations wererecorded. Table 2 shows IL-6 is among the genes that were noted toconsistently correlate with CXCL1 expression, others include Jagged 1protein (JAG1) and Chondromodulin-1 (LECT1). A Tumor Metastasis PCRarray containing 84 targets was also queried from two independentexperiments and significant fold change deviations (p<0.05) wererecorded. Table 2 shows TIMP4 is among the genes that were noted toconsistently correlate with CXCL1 expression, others includeInsulin-like growth factor 1 (IGF1) and Matrix metalloproteinase 2(MMP2).

TABLE 2 Fold change angiogenesis- or metastasis-related genes in PCRarrays T24 DU145 PC3 (vs. shSCR) (vs. Empty) (vs. shSCR) CXCL1 CXCL1CXCL1 PCR array Genes KD4 OE8 KD7 Angiogenesis Interleukin 6 (IL6) 0.41± 0.08  3.2 ± 0.19 0.52 ± 0.05 array Jagged 1 protein 0.49 ± 0.11  3.4 ±0.16 0.61 ± 0.09 (PAHS- (JAG1) 024Z) Chondromodulin-1 6.13 ± 0.24 1.23 ±0.19 3.94 ± 0.18 (LECT1) † Tumor Insulin-like growth 0.49 ± 0.07  2.6 ±0.29 0.59 ± 0.11 Metastasis factor 1 (IGF1) array matrix metallo- 0.04 ±0.02  1.0 ± 0.16 0.45 ± 0.11 (PAHS- proteinase 028Z) 2 (MMP2)Metalloproteinase 2.11 ± 0.21 0.22 + 0.04 1.52 ± 0.30 inhibitor 4(TIMP4) ‡ † Anti-angiogenic factor ‡ inhibitor of tumor invasion

This mechanistic finding of this study is that CXCL1 inducesangiogenesis and invasion by regulating IL-6 and TIMP4, respectively.TIMP4 is a member of the tissue inhibitors of metalloproteinases (MMPs)family, which is comprised of four members (TIMP1-4) with high sequencehomology and structural identity, but with different tissue expression,regulation and inhibitory characteristics. The TIMPs regulate suchdiverse processes as extracellular matrix (ECM) remodeling, and growthfactors and their receptors' activities through the inhibition of MMPs.Numerous tumors, including bladder and prostate, have been noted to havelower levels of TIMP4 (Melendez-Zajgla et al, 2008). IL-6 is amultifunctional pro-inflammatory cytokine that functions in inflammationand the maturation of B cells. IL-6 expression and function are alteredin inflammatory-associated disease states (e.g., arthritis) as well asin several human cancers, including prostate (Culig, 2014) and bladdercancer (Chen et al, 2013). Binding of IL-6 to its membrane receptor isfollowed by initiation of signal transduction through one of severalpathways: JAK/STAT, MAPK and/or PI3K pathways. In addition to regulationthrough its membrane receptor, IL-6 also acts through trans-signaling inregulation of proliferation, migration, and invasion (Santer et al,2010). Thus, these results suggest that CXCL1 influences tumor growththrough a) the induction of IL-6, which leads to enhancement of cellularproliferation, migration and invasion and b) the inhibition of TIMP4 mayfacilitate the activation of MMPs further enabling cellular growth andmotility, while therapeutically targeting CXCL1 inhibits these moleculesand halts these processes.

The apoptotic index in xenografts was evaluated using cleaved caspase-3immunostaining. Analyses revealed a significant increase in cleavedcaspase-3 (indication of apoptosis) in both T24 and PC3 xenograftstreated with 8 mg/kg of HL2401.

FIGS. 6A and 6B show that apoptotic index was increased in T24 by 35%(p<0.05) in tumors from animals treated with 8 mg/kg of HL2401.Apoptotic index in PC3 xenograft tumors was increased by 42% (p<0.05) intumors treated with 8 mg/kg of HL2401. To monitor associated angiogenicindex in these xenografts, microvessel density (MVD) was evaluated usingPECAM-1 immunostaining. Analyses revealed a significant reduction of MVD(angiogenesis) in both T24 and PC3 xenografts treated with 8 mg/kg ofHL2401. MVD was reduced in T24 by 52% (p<0.05) and by 43% (p<0.05) inPC3 tumors from animals treated with 8 mg/kg of HL2401.

To evaluate associated proliferative capability in these xenografts,proliferation index was evaluated using Ki-67 immunostaining. FIGS. 6Aand 6B show, in line with the observed reduction in MVD, a reduction inproliferation index was evident in both T24 and PC3 xenografts treatedwith 8 mg/kg of HL2401. Proliferative index was reduced in T24 by 50%(p<0.05) and by 39% (p<0.05) in tumors from animals treated with 8 mg/kgof HL2401. These in vivo observations corroborate the in vitro findingsand confirm a role for CXCL1 regulation of tumor growth associated withan increase in IL-6 expression and a reduction in TIMP4 expression, andsupport a role for CXCL1 as well as a role as a viable therapeutictarget.

These results show administration of a neutralizing antibody thattargets CXCL1, such as HL2401, resulted in the inhibition of endothelialsprouting, the inhibition of cellular invasion and the diminution ofbladder and prostate xenograft growth through the inhibition ofangiogenesis and proliferation, and the induction of apoptosis (REF).Therefore, therapeutic targeting of the chemokine CXCL1 could offer anovel strategy to inhibit tumor establishment and growth.

Furthermore, CXCL1, an inflammatory chemokine, may lead to therecruitment of inflammatory cells, such as lymphocytes and neutrophils.It is known that expression of the CXCL1 gene is accompanied byneutrophil infiltration. Therefore, anti-CXCL1 mAb of the presentdisclosure may be used to treat various conditions, such as cancer andother proliferative or inflammatory diseases.

In sum, CXCL1 expression in human cancer epithelial cells stimulatescells to invade and stimulates sprouting of endothelial cells. Inaddition, anti-CXCL1 neutralizing monoclonal antibody (HL2401) of thepresent disclosure can: a) inhibit cellular proliferation, b) inhibitcellular invasion, c) inhibit endothelial sprouting, and d) result inthe inhibition of subcutaneous xenograft tumors expressing CXCL1 viareduction in both proliferation and angiogenesis, and the induction ofapoptosis. Subsequently, the CXCL1 expression is positively correlatedwith the expression of IL-6 and inversely correlated with TIMP4expression.

Example 6 Humanization of HL2401 Clone A Humanized Single Chain VariableFragment (scFv) Antibody (Hum HL2401_scFv)

It is common in the field of recombinant humanized antibodies to graftmurine CDR sequences onto a well-established human immunoglobulinframework previously used in human therapies such as the frameworkregions of Herceptin [Trastuzumab]. In the construction of the humanScFv disclosed in this study a novel approach was taken to engineer aunique human immunoglobulin framework in order to avoid previousintellectual property issues surrounding the Herceptin framework[Genentech]. The humanized ScFv disclosed is thus anticipated toconstitute a distinct patentable composition of matter.

The design strategy for this human ScFv antibody was to engineer optimalhuman consensus sequences for each of the variable heavy chain and lightchains framework regions. This genetic engineering was achieved by firstidentifying human immunoglobulin germline genes orthologous to themurine heavy and light chain genes that comprise the murine mAbHL2401_scFv. Through analysis of human germline genes, a human consensussequence was then designed that constituted a minimal positionaltemplate and afforded optimal chain packing residues of sufficientlength to maintain overall 3-D conformation of the critical CDRresidues. The template of this human consensus sequence was predicted,based on spacing and topological considerations, to retain the bindingproperties of the original mouse monoclonal antibody. The humanconsensus gene that was engineered, Hum HL2401_scFv, was predicted toencode an immunoglobulin sequence most similar in sequence and toreproduce the three-dimensional protein conformation and chargeorientations within the paratope of the original ‘parent” murineHL2401_scFv sequence.

As a key design strategy for the creation of this immunochemicallyactive human ScFv that retains immunoreactivity with its cognateantigen, the approach outlined above may constitute a patentable methodor process in its own right. The genetic engineering strategy forcreating humanization murine monoclonal antibodies is thus based onselected germlines sequences that originate from un-rearrangedimmunoglobulin genes, based on the assumption that such frameworksshould therefore be free from idiosyncratic mutations and are minimallyimmunogenic. Coupled with design tools that permit 3-D homologymodelling of the resultant humanized ScFvs, comparisons can be made ofthe predicted humanized ScFv with the mouse monoclonal antibody proteinstructures and optimal CDR conformations can be maintained by editingand reshaping the variable region through varying the selection ofpacking residues at the interface of VH/VL. For patent disclosurepurposes this genetic engineering strategy may constitute process claimsin addition to claims covering composition of matter.

In this disclosure, the design of framework and complementaritydetermining regions of the humanized scFv Hum HL2401_scFv are outlined,including the 15-amino acid serine/glycine linker arm joining the heavyand light chains; the humanized ScFv is modelled in three dimensions;the purification of the ScFv is documented, and the specificimmunoreactivity of the ScFv Hum HL2401_scFv with human CXCL1 will bedetermined.

General Approach

The three CDR regions of the HL2401 murine heavy chain [H-CDR₁, H-CDR₂and H-CDR₃] and the three CDR regions of the murine light chain [L-CDR₁,L-CDR₂, L-CDR₃] were grafted onto optimized human immunoglobulin heavyand light chain frameworks.

Antibody Numbering Scheme and CDR Definitions

The antibody-numbering server that is part of the KabatMan databasehttp://www.bioinf.org.uk/) was used to number all antibody sequences inthis study according to the enhanced Chothia scheme. In the humanizationstrategy, we have combined the enhanced Chothia numbering scheme withthe contact CDR definition of antibody sequence to position the CDRs ofthe murine antibody light chain and heavy chains at the followinglocations: H-CDR₁ 26-35, H-CDR₂ 47-65, H-CDR₃ 93-101, L-CDR₁ 24-36,L-CDR₂ 46-55, and L-CDR₃ 89-96.

Selection of the Germline Based Human Consensus Template

To generate a humanized ScFv gene, six complementary determine regions(CDRs) of mouse VH and VL were grafted onto selected germlines basedhuman consensus frameworks (FRs) showing the highest amino acidssequence identity to optimize the humanization and thus the predictedimmunogenicity of the resultant ScFv protein. Human immunoglobulingermlines sequence showing the highest amino acid sequences similarityin FRs between human and mouse Hum HL2401 VH and VL were identifiedindependently using from VBASE2-quest serverhttp://www.vbase2.org/V-base (http://www.imgt.org/IMGT_vquest) andIg-BLAST server (http://www.ncbi.nlm.nih.gov/igblast). The highest fourconserved human germline immunoglobulin sequences for heavy chain andlight chains were selected. From these four human germlineimmunoglobulin sequences consensus human frameworks were designed forthe grafting of CDRs residues of the “parent” mouse HL2401_scFv. Theamino acid sequences in FRs of mouse VH and VL that differed fromgermline-based consensus human FRs were substituted with appropriatehuman residues, while preserving mouse residues at position known asVernier zone residues and chain packing residues. Important to theconstruction of this biologically active human ScFv was the substitutionof appropriate human residues in the framework regions whenever murinesequences differed from consensus human framework sequences. Thisincluded human sequences considered “Vernier zone residues” as well aschain packing residues.

Table 3 summarizes antibody gene after humanization.

TABLE 3 Z value Z value Gene sequence (humanness of VH) (humanness ofVL) Mouse Gene −1.4 −0.6 (MumHL2401 scFv) HumBB2401 scFv −0.5 0.7humanized version 1 HUM2401-ScFv_1 0.8 0.7 humanized version 2

TABLE 4 Humanized Version Domain Protein sequence DNA sequence HumBB2401scFv VL SEQ ID NO: 12 SEQ ID NO: 13 (humanized VH SEQ ID NO: 14 SEQ IDNO: 15 version 1) Full length SEQ ID NO: 16 SEQ ID NO: 17 HUM2401-ScFv_1VL SEQ ID NO: 12 SEQ ID NO: 13 humanized VH SEQ ID NO: 18 SEQ ID NO: 19version 2 Full length SEQ ID NO: 20 SEQ ID NO: 21

FIG. 7 shows homology modelling of mouse and humanization templatesequence using Rosetta homology modelling server. Green color schematicmodel indicated for mouse sequence template and firebrick colorschematic model indicated HumBB2401 scFv (humanized version 1). Thediagram was generated using PyMole molecular graphic system.

Second Strategy

HUM2401-ScFv_1 (humanized version 2) was generated by fusing HumBB2401scFv VL (SEQ ID NO: 12) with VH (SEQ ID NO: 18).

FIG. 8 shows homology modelling of mouse and humanization templatesequence using Rosetta homology modelling server. Green color schematicmodel indicated for mouse sequence template and firebrick colorschematic model indicated HUM2401-ScFv_1 (humanized version 2). Thediagram was generated using PyMole molecular graphic system.

FIG. 9 shows sequence alignment between mouse gene, MumHL2401 scFv andhumanized genes, i.e., HumBB2401 scFv (humanized version 1) andHUM2401-ScFv_1 (humanized version 2).

Additional humanized clones are shown in Table 5.

TABLE 5 Humanized Version Protein sequence DNA sequence Hum2401 scFv-3SEQ ID NO: 22 SEQ ID NO: 23 HumBB2401-3 SEQ ID NO: 24 SEQ ID NO: 25

FIG. 10 shows sequence alignment between Hum2401 scFv-3 and HumBB2401-3.

Induction of ScFv Proteins in Bacterial Host

The humanized HL2401_scFv clones were constructed into bacterialexpression vector and transformed into T7 shuffle bacterial strain. T7shuffle cells and was grown in 1.5 L 2×YT plus ampicillin medium at 37°C. until log-phage (OD600=0.5), induced with 0.3 mM IPTG, and allowed togrow at 30° C. for an additional 16 hrs. After induction, the bacteriawere harvested by centrifugation at 8000 g for 15 min at 4° C., and thepellets were stored in −20° C. for at least 2 hrs. The frozen pelletswere briefly thawed and suspended in 40 ml of lysis buffer (1 mg/mllysozyme in PBS plus EDTA-free protease inhibitor cocktail (ThermoScientific, Waltham, Mass.). The lysis mixture was incubated on ice foran hour, and then 10 mM MgCL2 and 1 μg/ml DNase I were added and themixture was incubated at 25° C. for 20 min. The final lysis mixture wascentrifuged at 12000 g for 20 min and the supernatants were collected.This supernatant was termed the periplasmic extract used for Protein Lcolumn affinity chromatography.

Western Blots Analysis Using HL2401_scFv Protein

Purified recombinant human CXCL1 protein was used as antigen target inWestern blot analyses. 500 ng human CXCL1 protein and 1 μg purifiedprotein as negative control were loaded onto 4-20% gradient Tris-glycineSDS-PAGE and transferred onto intracellular membranes. The membrane wasblocked using 3% skimmed milk in PBS for 3 h at room temperature. Afterthat, the membrane was incubated with partial purified humanizedHL2401_scFv protein overnight at 4° C. The membrane was washed withsodium phosphate buffered saline with 0.05% tween 20 buffer (PBST) 3times. The washed membrane was incubated with anti-c Myc mouse IgG for 1h at room temperature to recognize the c-Myc tag on the scFv andidentify the position of antigens bound by the scFv. After washing withPBST, the membrane was incubated with the goat anti-mouse IgG (H+L) HRPconjugate diluted (1:3000 v/v) in PBS for 1 h at RT, and specificimmunoreactive bands were visualized with a mixture of TMB substrate.

FIG. 11 shows the antigen CXCL1 and a negative control protein waselectrophoresed and transblotted to the nitrocellulose membrane. Theantigens were probed with the humanized version of scFvs antibodies,followed by anti-C Myc mouse monoclonal antibody and a respectivesecondary antibody conjugated to HRP. Bound antibodies were visualizedby using TMB. Lane M indicated for molecular marker, Lane 1 indicatesfor human CXCL1 protein (300ng) and lane 2 indicates as negativecontrol. Panel A represents for ponceau staining, Panel B representsWestern blot analysis using purified Hum2401 scFv antibody and Panel Crepresents Western blot analysis using purified HumBB2401 scFv antibody.

On the other hand, an anti-Myc tag monoclonal antibody, used torecognize the Myc tag on the expressed of scFv protein. The antigenloaded membrane was incubated with anti-myc-HRP ( 1/2000) antibody andspecific immunoreactive bands were visualized with a mixture of TMBsubstrate.

FIG. 12 shows humanized version of HL2401_scFv protein expressed in E.coli and purified that was detected by anti-His tag antibody.

ELISA Test for Confirm the Binding Activity of HL2401_scFv Protein

The human CXCL1 protein was coated onto 96-well, 30 ng for well at 4° C.overnight. The plate was blocked by 3% skim milk in PBS 2 h at roomtemperature. The plate was washed 3 times with PBST and appliedanti-human CXCL1 humanized version scFv antibodies at different dilutionconcentration. The anti-Myc mouse monoclonal antibody with HRP conjugateantibody was applied and developed with TMB solution.

FIG. 13 shows the humanization version of H2407 scFvs format antibodyproteins are specially binding to human CXCL1 antigen in ELISA andWestern blot analysis. This ELISA and Western blot analysis results donot correlate as dose depend activity of these antibody.

Example 7 Design the Reformatting Hum2401 Derivatives

Light chain of both humanize version may be fused with a signal peptide:

(SEQ ID NO: 26) MDSQAQVLMLLLLWVSGTCG.

Heavy chain of humanize version Hum2401 derivatives may be fused with asignal peptide: MEFGLSWVFLVAILKGVQC (SEQ ID NO: 27).

The fusion proteins of the Hum2401 derivatives are summarized in Table6.

TABLE 6 Hum2401 fusion protein Protein sequence DNA sequence Hum2401VL-signal SEQ ID NO: 28 SEQ ID NO: 29 Hum2401 VH-signal SEQ ID NO: 30SEQ ID NO: 31 HumBB 2401 VH-signal SEQ ID NO: 32 SEQ ID NO: 33

Sub-Cloning into Mammalian Expression Vector

As shown in FIG. 14, the light chain and heavy chain of humanized genewere sub-cloned using EcoR I and Apa I for heavy chain and Hind III andBsiWi for light chain. First, the light chain gene was cloned intovector and plasmid DNA was sequenced. The correct light chain insertedclone was used for second step sub-cloning for heavy chain of twoversion of humanization. The ligated clones were amplified and send forsequencing. The correct inserted heavy chain clones were used for largescale DNA preparation. Preparation of large amount of plasmid DNA usingendotoxin free Kit. The clones are summarized in Table 7.

TABLE 7 Clone Protein sequence DNA sequence PCMV-dhfr-H2401 SEQ ID NO:34 SEQ ID NO: 35 Light chain clone 5 Pcmv-dhfrhumBB2401 SEQ ID NO: 36SEQ ID NO: 37 VH clone 1 Pcmv dhfr- SEQ ID NO: 38 SEQ ID NO: 39HumBB2401 clone 4

Cell Line HEK293 F Suspension Culture Transient Transfection andProduction in Suspension HEK 293-F Cells

1. Approximately 24 hours before transfection, pass Freestyle 293-Fcells at 0.6×10⁶-0.7×10⁶ cells/mL. Place the flasks (125 mL or 100 mLErlenmeyer flasks with ventilation membrane caps) on an orbital shakerplatform rotating at 135 rpm at 37° C., 8% CO2.

2. On the day of transfection, the cell density should be about1.2×10⁶-1.5×10⁶/mL. Dilute the cells to 1×10⁶ cells/mL. Add 30 mL of thecells into each 125-mL shake flask.

-   -   a. For small scale productions in Erlenmeyer flasks containing        100 mL total working volume of cell suspension incubated at 150        rpm in a linear shaker.    -   b. A total of 1 μg high quality plasmid-DNA (prepared using        Takara Clontech Nucleobond Xtra Midi EF plasmid isolation kit)        and 2.5 μg PEI per mL culture volume (total 100 μg DNA) was        prepared in 1/10 volume of fresh serum free culture medium.    -   c. Dilute PEI in appropriate volume of serum free medium in a        polystyrol Plate or tube (Do not use polypropylene tubes).    -   d. Dilute Plasmid-DNA in appropriate volume DMEM and mix with        the PEI Suspension.    -   e. Incubate the mixture at RT for 30 min to allow formation of        PEI::DNA complexes.    -   f. Disperse PEI::DNA suspension evenly over the cells.    -   g. Cells are further cultured for 6-day.    -   h. Test yield of human IgG.

Preparation of the Transfection Reagent, PEI

PEI (polyethylenimine), a cationic polymer is a 25 kDa linear fromPolysciences (Polysciences, Cat. No. 23966-2) Note: Portolano et al(2014) used branched form of PEI (Sigma Aldrich cat. No. 408727).

Assay for Activity of Humanized IgG Version ELISA Test for Confirm theBinding Activity of HL2401_scFv Protein

The human CXCL1 protein was coated onto 96-well, 30 ng for well at 4° C.overnight. The plate was blocked by 3% skim milk in PBS 2 h at roomtemperature. The plate was washed 3 times with PBST and appliedanti-human CXCL1 humanized version IgG antibodies were diluted 1/100 and1/10 in PBS and subjected into antigen coated plate for 1 h at roomtemperature. The donkey anti-human monoclonal antibody with HRPconjugate antibody was diluted 1/3000 and applied for 45 min at roomtemperature, afterwards washed 3 times with PBST and developed with TMBsolution.

Table 8 and FIG. 15 show the ELISA results.

TABLE 8 Hum2401 Ab ELISA signals Min 1/10 dilute original 2.098 2.0982.098 1/100 dilute original 1.848 1.908 1.848 only anti-Human HRP 0.2060.075 0.075 Blank 0.044 0.044 0.044 HumBB2401 Ab ELISA signals Min 1/10dilute original 2.091 2.094 2.091 1/100 dilute original 1.662 1.6651.662 only anti-Human HRP 0.151 0.121 0.121 Blank 0.043 0.048 0.043

Western Blots Analysis Using Humanized HL2401_IgG Derivatives

Purified recombinant human CXCL1 protein was used as antigen target inWestern blot analyses. 200 ng and 500 ng human CXCL1 protein andbacterial cell lysate with chicken lysozyme protein as negative controlwere loaded onto 4-20% gradient Tris-glycine SDS-PAGE and transferredonto intracellular membranes. The membrane was blocked using 3% skimmedmilk in PBS for 3 h at room temperature. After that, the membrane wasincubated with transfected supernatant of humanized HL2401_IgG variantsprotein overnight at 4° C. The membrane was washed with sodium phosphatebuffered saline with 0.05% tween 20 buffer (PBST) 3 times. The washedmembrane was incubated with donkey anti-Human (H+L) HRP conjugateantibody for 1 h at room temperature to recognize the humane heavy andlight chain of constant region and identify the position of antigensbound by the reformatting humanized antibodies, and specificimmunoreactive bands were visualized with a mixture of TMB substrate.

FIG. 16 shows the antigen human CXCL1 and a negative control bacterialcell lysate with chicken lysozyme protein were electrophoresed andtransblotted to the nitrocellulose membrane. The antigens were probedwith the humanized version of IgG antibodies, followed by anti-human(H+L) secondary antibody conjugated to HRP. Bound antibodies werevisualized by using TMB. Lane M indicated for molecular marker, Lane 1represents for human CXCL1 protein (200 ng) and lane 2 represents humanCXCL1 protein (200 ng) Lane 3 represents as negative control. Panel Arepresents for SDS-PAGE staining, Panel B represents Western blotanalysis using purified Hum2401 IgG antibody and Panel C representsWestern blot analysis using purified HumBB2401 IgG antibody,respectively.

Purification of Transfected Culture Supernatant Using Protein G affinityChromatography

The transfected cells were harvested and filter the supernatant using0.45 u and loaded protein G affinity chromatography using AKTA pure Lsystem. The protein was eluted using 0.2M Glycine pH2.5 and neutralizedwith 1M Tris-HCL pH: 9. the eluted fractions were analyzed intoSDS-PAGE.

FIGS. 17A and 17B show affinity purification chromatogram of Hum2401 andHumBB2401 IgG, respectively.

Table 9 summarized the affinity purification chromatogram results.

TABLE 9 Approx. Total Concentration Protein Concentration Volume mg/mlHum2401IgG 1.6 mg 3 ml 0.539 mg/ml HumBB2401   1 mg 3 ml  0.35 mg/ml

FIG. 18 shows SDS-PAGE gel electrophoresis of purified samples, Panel Arepresents for SDS-PAGE staining of purified protein Hum2401 peak 9 andPanel B represents for SDS-PAGE staining of purified protein Hum2401peak 10.

List of Deliverable Samples

1. Protein Samples

Approx. Total Concentration Sample name Concentration total Volume mg/mlHum2401IgG 1.6 mg 3 ml 0.539 mg/ml HumBB2401   1 mg 3 ml  0.35 mg/ml

2. Plasmid DNA Samples

Sample name Specification Plasmid Name Sample POE-mu H2401scFv MouseScFv expression vector Plasmid scFv under T7 promoter DNA (periplasmic)POE- Humanized ScFv expression vector Plasmid HumH2401scFv scFv under T7promoter DNA (periplasmic) POE- Humanized ScFv expression vector PlasmidHumBBH2401scFv scFv under T7 promoter DNA (periplasmic) Pcmvdhfr-Reformatting Mammalian expression Plasmid Hum2401(H + L) humanizedvector including DNA IgG version humanized gene Pcmvdhfr- ReformattingMammalian expression Plasmid HumBB2401 humanized vector including DNA(H + L) IgG version humanized gene

Example 8 Effects on Angiogenesis by Humanized Anti-CXCL1 Antibodies

Humanized anti-CXCL1 antibodies inhibit endothelial cell sprouting

FIGS. 19A-19H show that humanized anti-human CXCL1 antibodies BB2401 andHum2401 are as effective as Avastin in the inhibition of endothelialcell sprouting. Briefly, the positive control Bevacizumab (Avastin) atfrequently used concentration (2 mg/ml) significantly reduced number ofsprout (FIG. 19A) and total length of sprouts (FIG. 19E). Mouseanti-human CXCL1 antibody HL2401 and commercial anti-human CXCL1antibody (Anti-CXCL1) also significantly reduced number of sprout (FIGS.19B and 19C) and total length of sprouts (FIGS. 19F and 19G). The effectof anti-human CXCL1 antibodies BB2401 and Hum2401 were tested incomparison with Avastin at the concentration of 200 μg/ml. Avastin atthe concentration of 200 μg/ml showed similar level of inhibitoryeffects as 2 mg/ml on the reduction in number of sprout (FIG. 19D) andtotal length of sprouts (FIG. 19H). Hum2401 had the same level ofinhibitory effects as Avastin (FIGS. 19D and 19H). Notably, BB2401demonstrated stronger inhibitory effects than Avastin on number ofsprout (FIG. 19D) and total length of sprouts (FIG. 19H). Takentogether, neutralization of CXCL1 by humanized anti-human CXCL1antibodies BB2401 and Hum2401 leads to inhibition of angiogenesis. Theasterisks *, **, ***, and **** indicate the p value is 0.05, <0.01,<0.001 and <0.0001, respectively.

Humanized Anti-CXCL1 Antibodies Inhibit Endothelial Cell Tube Formation

FIGS. 20A-20F show that humanized anti-human CXCL1 antibodies BB2401 andHum2401 are as effective as Avastin in the inhibition of endothelialcell tube formation. Briefly, the positive control Avastin at frequentlyused concentration (1 mg/ml) significantly reduced total length oftubing (FIG. 20D), but not number of junction (FIG. 20A). As comparativecontrol, mouse anti-human CXCL1 antibody HL2401 and commercialanti-human CXCL1 antibody (anti-CXCL1) did not significantly reduce thenumber of junction (FIG. 20B) and total length of tubing (FIG. 20E). Theeffect of BB2401 and Hum2401 in comparison with Avastin at theconcentration of 100 μg/ml was tested. FIG. 20F shows Avastin at theconcentration of 100 μg/ml has a similar inhibitory effects on totallength of tubing as at the concentration of 1 mg/ml (FIG. 20D). FIG. 20Falso shows both BB2401 and Hum2401 had the same levels of inhibitoryeffects as Avastin on total length of tubing. The asterisks *, ** and*** indicate the p value is 0.05, <0.01 and <0.001, respectively.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the disclosureand does not pose a limitation on the scope of the disclosure unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of thedisclosure unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents.

The present disclosure includes all modifications and equivalents of thesubject matter recited in the aspects or claims presented herein to themaximum extent permitted by applicable law.

All references cited in this specification are herein incorporated byreference as though each reference was specifically and individuallyindicated to be incorporated by reference. The citation of any referenceis for its disclosure prior to the filing date and should not beconstrued as an admission that the present disclosure is not entitled toantedate such reference by virtue of prior disclosure.

It will be understood that each of the elements described above, or twoor more together may also find a useful application in other types ofmethods differing from the type described above. Without furtheranalysis, the foregoing will so fully reveal the gist of the presentdisclosure that others can, by applying current knowledge, readily adaptit for various applications without omitting features that, from thestandpoint of prior art, fairly constitute essential characteristics ofthe generic or specific aspects of this disclosure set forth in theappended claims. The foregoing embodiments are presented by way ofexample only; the scope of the present disclosure is to be limited onlyby the following claims.

What is claimed is:
 1. A method of treating a patent who has cancer,comprising administering to the patient an antibody comprising a heavychain variable domain comprising CDR1 comprising the amino acid sequenceof SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO:7, and CDR3 comprising the amino acid sequence of SEQ ID NO: 8, whereinthe heavy chain variable domain comprises at most one amino acidsubstitution in the CDR1, the CDR2, and/or the CDR3, and a light chainvariable domain comprising CDR1 comprising the amino acid sequence ofSEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 10,and CDR3 comprising the amino acid sequence of SEQ ID NO: 11, whereinthe light chain variable domain comprises at most one amino acidsubstitution in the CDR1, the CDR2, and/or the CDR3, wherein the cancerexpresses human chemokine CXCLI protein comprising the amino acidsequence of SEQ ID NO:
 5. 2. The method of claim 1, wherein the lightchain variable domain comprises the amino acid sequence of SEQ ID NO: 12and the heavy chain variable domain comprises the amino acid sequence ofSEQ ID NO: 14 or
 18. 3. The method of claim 1, wherein the light chainvariable domain comprises the amino acid sequence of SEQ ID NO: 28 andthe heavy chain variable domain comprises the amino acid sequence of SEQID NO: 30 or
 32. 4. The method of claim 1, wherein the light chainvariable domain comprises CDR regions CDR1, CDR2, and CDR3 of the aminoacid sequence of SEQ ID NO: 12, and the heavy chain variable domaincomprises CDR regions CDR1, CDR2, and CDR3 of the amino acid sequence ofSEQ ID NO: 14 or
 18. 5. The method of claim 1, wherein the light chainvariable domain comprises CDR regions CDR1, CDR2, and CDR3 of the aminoacid sequence of SEQ ID NO: 28, and the heavy chain variable domaincomprises CDR regions CDR1, CDR2, and CDR3 of the amino acid sequence ofSEQ ID NO: 30 or
 32. 6. The method of claim 1, wherein the heavy chainvariable domain comprises CDR1 consisting of the amino acid sequence ofSEQ ID NO: 6, CDR2 consisting of the amino acid sequence of SEQ ID NO:7, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 8, andthe light chain variable domain comprises CDR1 consisting of the aminoacid sequence of SEQ ID NO: 9, CDR2 consisting of the amino acidsequence of SEQ ID NO: 10, and CDR3 consisting of the amino acidsequence of SEQ ID NO:
 11. 7. The method of claim 1, wherein theantibody comprises the amino acid sequence of SEQ ID NO: 16, 20, 22, or24.
 8. The method of claim 1, wherein the light chain variable domaincomprises a signal peptide comprising the amino acid sequence of SEQ IDNO: 26 and the heavy chain variable domain comprises a signal peptidecomprising the amino acid sequence of SEQ ID NO:
 27. 9. The method ofclaim 1, wherein the cancer is bladder cancer or prostate cancer. 10.The method of claim 1, wherein the antibody is in the form of acomposition comprising an adjuvant, and optionally, a pharmaceuticallyacceptable carrier, and optionally, pharmaceutically acceptableexcipients and/or stabilizers.
 11. The method of claim 10, wherein theadjuvant is selected from the group consisting of anti-CD40 antibody,imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 12. The methodof claim 1, wherein the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 12, and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO:
 14. 13. The method ofclaim 1, wherein the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 12, and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO:
 18. 14. The method ofclaim 1, wherein the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 28, and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO:
 30. 15. The method ofclaim 1, wherein the light chain variable domain comprises the aminoacid sequence of SEQ ID NO: 28, and the heavy chain variable domaincomprises the amino acid sequence of SEQ ID NO:
 32. 16. The method ofclaim 1, wherein the antibody comprises the amino acid sequence of SEQID NO:
 16. 17. The method of claim 1, wherein the antibody comprises theamino acid sequence of SEQ ID NO:
 20. 18. The method of claim 1, whereinthe antibody comprises the amino acid sequence of SEQ ID NO:
 22. 19. Themethod of claim 1, wherein the antibody comprises the amino acidsequence of SEQ ID NO:
 24. 20. The method of claim 1, wherein theantibody binds to a human chemokine CXCL1 protein comprising the aminoacid sequence of SEQ ID NO: 5.